Crystalline pharmaceutical and methods of preparation and use thereof

ABSTRACT

Novel crystalline polymorphic forms, Forms A, B, C, D, and E of a compound of Formula I, which has been found to be a potent inhibitor of LFA-1, are disclosed. Methods of preparation and uses thereof in the treatment of LFA-1 mediated diseases are also disclosed in this invention.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.12/909,241, filed Oct. 21, 2010 which claims priority from U.S.Provisional Application 61/253,828, filed Oct. 21, 2009, whichapplications are hereby incorporated by reference in their entirety.

BACKGROUND OF THE INVENTION

The compound of Formula I:

has been found to be an effective inhibitor of LymphocyteFunction-Associated Antigen-1 (LFA-1) interactions with the family ofIntercellular Adhesion Molecules (ICAM), and has desirablepharmacokinetic properties, including rapid systemic clearance. Improvedforms, including crystalline forms, and their uses in treatment ofdisorders mediated by the interaction of LFA-1 and ICAM are describedherein. Novel polymorphs of the compound of Formula I which may affordimproved purity, stability, bioavailability and other likecharacteristics for use in pharmaceutical formulations and methods ofuse thereof are useful in treating disease.

SUMMARY OF THE INVENTION

In a first aspect the invention provides methods of making a compound ofFormula I:

According to the invention, such methods may yield a compound whichcomprises a purity of greater than about 90%, and its pharmaceuticallyacceptable salts. In some embodiments, the purity is greater than about98%. In other embodiments, the compound comprises at least about 95% ofan S-enantiomer. In yet other embodiments, the compound is not thecalcium salt of the free acid. Alternatively, the compound is a sodiumsalt.

The invention also provides compositions of a compound of Formula I,wherein the compound comprises less than 0.5% of any one byproduct ofchemical synthesis of the compound. In other embodiments, the compoundcomprises less than a total of 1.5% of all byproducts of the chemicalsynthesis. In yet other embodiments, the compound comprises less than atotal of 1.5% of all byproducts of the chemical synthesis.

In a second aspect of the invention, a method of synthesizing a compoundof Formula I is provided:

comprising the steps:

-   -   a) base hydrolysis of Formula AA:

-   -   with a base in an aprotic solvent; and    -   b) isolating a compound of Formula I. In some embodiments, the        aprotic solvent is dioxane. In some embodiments, the base is a        strong base, e.g., sodium hydroxide. In various embodiments, R        is a carbon containing moiety.

In a third aspect of the invention, a method of synthesizing a compoundof Formula I is provided:

-   -   comprising the steps:    -   a) base hydrolysis of Formula A with a base in an aprotic        solvent; and

-   -   b) isolating a compound of Formula I. In some embodiments, the        aprotic solvent is dioxane. In some embodiments, the base is a        strong base, e.g., sodium hydroxide.

In a fourth aspect of the invention, a method of synthesizing a compoundof Formula I is provided comprising the steps of (a) acid hydrolysis ofFormula AA or Formula A with an acid in an aprotic solvent; and (b)isolating a compound of Formula I. In some embodiments, the aproticsolvent is dioxane. In some embodiments, the acid is a strong acid,e.g., hydrogen chloride. In various embodiments, R is a carboncontaining moiety.

In a fifth aspect of the invention, the invention is directed to acompound of Formula I synthesized according to the methods disclosedherein. The compound of Formula I may be isolated, and in variousembodiments, the compound of Formula I synthesized according to themethods of the invention has an enantiomeric excess of greater thanabout 98%.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication, patent, or patent application wasspecifically and individually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the invention are set forth with particularity inthe appended claims. A better understanding of the features andadvantages of the present invention will be obtained by reference to thefollowing detailed description that sets forth illustrative embodiments,in which the principles of the invention are utilized, and theaccompanying drawings of which:

FIG. 1 is a graphical representation of the FT-Raman spectrum of theamorphous form of the compound of Formula I.

FIG. 2 is a graphical representation of the PXDR pattern of theamorphous form of the compound of Formula I.

FIG. 3 is a graphical representation of the TG-FTIR chromatogram of theamorphous form of the compound of Formula I.

FIG. 4 is a graphical representation of the FT-Raman spectrum of theamorphous form of the compound of Formula I overlaid for comparison withthe FT-Raman spectrum of crystalline Form A.

FIG. 5 is a graphical representation of the FT-Raman spectrum ofcrystalline Form A.

FIG. 6 is a graphical representation of the PXDR pattern of crystallineForm A.

FIG. 7 is a graphical representation of the TG-FTIR chromatogram ofcrystalline Form A.

FIG. 8 is a graphical representation of the DSC chromatogram ofcrystalline Form A.

FIG. 9 is a graphical representation of the DVS chromatogram ofcrystalline Form A with time as the X axis.

FIG. 10 is a graphical representation of the DVS chromatogram ofcrystalline Form A with relative humidity as the X axis.

FIG. 11 is a graphical representation of a TG-FTIR chromatogram ofcrystalline Form A, using material previously subjected to DVS analysis.

FIG. 12 is a graphical representation of the FT-Raman spectrum ofcrystalline Form B.

FIG. 13 is a graphical representation of the PXDR pattern of crystallineForm B.

FIG. 14 is a graphical representation of the TG-FTIR chromatogram ofcrystalline Form B.

FIG. 15 is a graphical representation of the DVS chromatogram ofcrystalline Form B with time as the X axis.

FIG. 16 is a graphical representation of the DVS chromatogram ofcrystalline Form B with relative humidity as the X axis.

FIG. 17 is a graphical representation of the DSC chromatogram ofcrystalline Form B.

FIG. 18. is a graphical representation of the FT-Raman spectrum ofcrystalline Form C.

FIG. 19 is a graphical representation of the PXDR pattern of crystallineForm C.

FIG. 20 is a graphical representation of the TG-FTIR chromatogram ofcrystalline Form C.

FIG. 21 is a graphical representation of the FT-Raman spectrum ofcrystalline Form D.

FIG. 22 is a graphical representation of the PXDR pattern of crystallineForm D.

FIG. 23 is a graphical representation of the TG-FTIR chromatogram ofcrystalline Form D.

FIG. 24 is a graphical representation of the FT-Raman spectrum ofcrystalline Form E.

FIG. 25 is a graphical representation of the PXDR pattern of crystallineForm E.

FIG. 26 is a graphical representation of the TG-FTIR chromatogram ofcrystalline Form E.

FIG. 27 is a graphical representation of histopathological evaluation ofbiopsies taken before and after treatment of a dog eye with the compoundof Formula I.

FIG. 28 illustrates the mean change in Schirmer test score at weeks, 2,4, 8, and 12 for eyes in dogs treated with the compound of Formula I.

FIG. 29 illustrates percentage of dog eyes with a Schirmer test score ofgreater than 10 mm at 2, 4, 8, and 12-weeks with a formulation of 1%compound of Formula I (TID; three times daily).

FIG. 30 illustrates percentage of eyes with a greater than 4 mmimprovement in Schirmer test score at 2, 4, 12, 16, and 26 weeks forsubjects treated with a formulation of 1% compound of Formula I (TID)compared to literature results for 2% CsA (BID; two times daily).

FIG. 31 illustrates a timecourse of mean plasma levels of the compoundof Formula I treatment (human) with 5% compound of Formula I.

FIG. 32 illustrates tear C_(min) levels for human subjects treated with1% compound of Formula I QD (once a day).

FIG. 33 illustrates the dose/drug C_(max) tear level relationship foradministration of the compound of Formula I in humans (QD and TID).

FIG. 34 illustrates the dose/AUC and dose/mean C_(max) tear levelrelationship for human subjects treated QD with the compound of FormulaI.

FIG. 35 is a graphical representation of a whole body autoradiograph fora male Sprague Dawley Animal 0.5 hour after a single topical ocularadministration of [¹⁴C]-compound of Formula I (1 mg/eye).

FIG. 36 is a graphical representation of a whole-body autoradiograph fora male Sprague Dawley Animal 2 hours after a single topical ocularadministration of [¹⁴C]-compound of Formula I (1 mg/eye).

FIG. 37 is a graphical representation of a whole-body autoradiograph fora male Sprague Dawley Animal 8 hours after a single topical ocularadministration of [¹⁴C]-compound of Formula I (1 mg/eye).

FIG. 38 is a graphical representation of a whole-body autoradiograph fora male Sprague Dawley Animal 12 hours after a single topical ocularadministration of [¹⁴C]-compound of Formula I (1 mg/eye).

FIG. 39 is a graphical representation of a whole-body autoradiograph fora male Sprague Dawley Animal 24 hours after a single topical ocularadministration of [¹⁴C]-compound of Formula I (1 mg/eye).

FIG. 40 illustrates rat ocular pharmacokinetics of [¹⁴C]-compound ofFormula I.

FIG. 41 illustrates dog ocular pharmacokinetics of [¹⁴C]-compound ofFormula I.

FIG. 42 is a graphical representation of the timecourse of drug plasmalevels for the compound of Formula I following single IV doses in rats.

FIG. 43 is a graphical representation of the timecourse of drug plasmalevels for the compound of Formula I following single IV doses in dogs.

FIG. 44 illustrates the dose/drug AUC (in tears) relationship for thecompound of Formula I administered to dogs.

FIG. 45 illustrates the drug tear concentration profiles measured after13 weeks of TID ocular dosing in rabbits.

FIG. 46 illustrates the drug tear concentration profiles measured after13 weeks of TID ocular dosing in dogs.

FIG. 47 illustrates mean drug tear concentrations in right and left eyesof rabbits following topical instillation of a single dose of thecompound of Formula I.

FIG. 48 illustrates the drug plasma level in rats for various topicalapplications of the compound of Formula I.

DETAILED DESCRIPTION OF THE INVENTION

While selected embodiments of the present invention have been shown anddescribed herein, it will be obvious to those skilled in the art thatsuch embodiments are provided by way of example only. Numerousvariations, changes, and substitutions will now occur to those skilledin the art without departing from the invention. It should be understoodthat various alternatives to the embodiments of the invention describedherein may be employed in practicing the invention. It is intended thatthe appended claims define the scope of the invention and that methodsand structures within the scope of these claims and their equivalents becovered thereby.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as is commonly understood by one of skill in theart to which this invention belongs. All patents and publicationsreferred to herein are incorporated by reference.

As used in the specification and claims, the singular form “a”, “an” and“the” includes plural references unless the context clearly dictatesotherwise.

The terms “antagonist” and “inhibitor” are used interchangeably, andthey refer to a compound having the ability to inhibit a biologicalfunction of a target protein, whether by inhibiting the activity orexpression of the target protein. Accordingly, the terms “antagonist”and “inhibitors” are defined in the context of the biological role ofthe target protein. While preferred antagonists herein specificallyinteract with (e.g. bind to) the target, compounds that inhibit abiological activity of the target protein by interacting with othermembers of the signal transduction pathway of which the target proteinis a member are also specifically included within this definition. Apreferred biological activity inhibited by an antagonist of LFA-1, forexample, is associated with an undesired inflammatory or immune responseas manifested in inflammatory or autoimmune disease, respectively.

A “directly competitive inhibitor” or “directly competitive antagonist”refers to a ligand, which includes biomolecules, peptides, and syntheticsmall organic molecules, which binds directly to the active site of thebiological target molecule, and directly prevents a substrate frombinding to it. For example, a directly competitive inhibitor of theinteraction of LFA-1 and ICAM-1, binds to LFA-1 at the site where ICAM-1binds, and thus directly prevents ICAM-1 from binding.

“Allosteric inhibitor” as used herein refers to a ligand which includesbiomolecules, peptides, and synthetic small organic molecules, thatbinds to a biological target molecule at a site other than the bindingsite of the interaction which is being inhibited. The interactionchanges the shape of the biological target molecule so as to disrupt theusual complex between the biological target molecule and its substrate.This results in inhibition of the normal activity of such complexformation. For example, an allosteric inhibitor of the interaction ofLFA-1 and ICAM-1, binds to LFA-1 at a site other than that where ICAM-1binds, but it disrupts the binding site of ICAM-1 such that theinteraction of LFA-1 and ICAM-1 is reduced.

The term “selective inhibition” or “selectively inhibit” as applied to abiologically active agent refers to the agent's ability to selectivelyreduce the target signaling activity as compared to off-target signalingactivity, via direct or interact interaction with the target.

The term “co-administration,” “administered in combination with,” andtheir grammatical equivalents, as used herein, encompassesadministration of two or more agents to an animal so that both agentsand/or their metabolites are present in the animal at the same time.Co-administration includes simultaneous administration in separatecompositions, administration at different times in separatecompositions, or administration in a composition in which both agentsare present.

The term “effective amount” or “therapeutically effective amount” refersto that amount of a compound described herein that is sufficient toeffect the intended application including but not limited to diseasetreatment, as defined below. The therapeutically effective amount mayvary depending upon the intended application (in vitro or in vivo), orthe subject and disease condition being treated, e.g., the weight andage of the subject, the severity of the disease condition, the manner ofadministration and the like, which can readily be determined by one ofordinary skill in the art. The term also applies to a dose that willinduce a particular response in target cells, e.g. reduction of plateletadhesion and/or cell migration. The specific dose will vary depending onthe particular compounds chosen, the dosing regimen to be followed,whether it is administered in combination with other compounds, timingof administration, the tissue to which it is administered, and thephysical delivery system in which it is carried.

As used herein, “treatment” or “treating,” or “palliating” or“ameliorating” are used interchangeably herein. These terms refers to anapproach for obtaining beneficial or desired results including but notlimited to therapeutic benefit and/or a prophylactic benefit. Bytherapeutic benefit is meant eradication or amelioration of theunderlying disorder being treated. Also, a therapeutic benefit isachieved with the eradication or amelioration of one or more of thephysiological symptoms associated with the underlying disorder such thatan improvement is observed in the patient, notwithstanding that thepatient may still be afflicted with the underlying disorder. Forprophylactic benefit, the compositions may be administered to a patientat risk of developing a particular disease, or to a patient reportingone or more of the physiological symptoms of a disease, even though adiagnosis of this disease may not have been made. The compositions maybe administered to a subject to prevent progression of physiologicalsymptoms or to prevent progression of the underlying disorder.

A “therapeutic effect,” as that term is used herein, encompasses atherapeutic benefit and/or a prophylactic benefit as described above. Aprophylactic effect includes delaying or eliminating the appearance of adisease or condition, delaying or eliminating the onset of symptoms of adisease or condition, slowing, halting, or reversing the progression ofa disease or condition, or any combination thereof.

As used herein, the term “pharmaceutically acceptable salt” refers tothose salts which are suitable for pharmaceutical use, preferably foruse in the tissues of humans and lower animals without undue irritation,allergic response and the like. Pharmaceutically acceptable salts ofamines, carboxylic acids, and other types of compounds, are well knownin the art. For example, S. M. Berge, et al., describe pharmaceuticallyacceptable salts in detail in J Pharmaceutical Sciences, 66: 1-19(1977), incorporated herein by reference. The salts can be prepared insitu during the final isolation and purification of the compounds of theinvention, or separately by reacting a free base or free acid functionwith a suitable reagent, as described generally below. For example, afree base function can be reacted with a suitable acid. Furthermore,where the compounds of the invention carry an acidic moiety, suitablepharmaceutically acceptable salts thereof may, include metal salts suchas alkali metal salts, e. g. sodium or potassium salts; and alkalineearth metal salts, e. g. calcium or magnesium salts. Examples ofpharmaceutically acceptable, nontoxic acid addition salts are salts ofan amino group formed with inorganic acids such as hydrochloric acid,hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid orwith organic acids such as acetic acid, oxalic acid, maleic acid,tartaric acid, citric acid, succinic acid or malonic acid or by usingother methods used in the art such as ion exchange. Otherpharmaceutically acceptable salts include adipate, alginate, ascorbate,aspartate, benzoate, bisulfate, borate, butyrate, camphorate,camphorsulfonate, citrate, cyclopentanepropionate, digluconate,dodecylsulfate, formate, fumarate, glucoheptonate, glycerophosphate,gluconate, hernisulfate, heptanoate, hexanoate, hydroiodide,2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, laurylsulfate, malate, maleate, malonate, methanesulfonate, nicotinate,nitrate, oleate, oxalate, palmitate, pectinate, persulfate,3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate,succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate,undecanoate, valerate salts, and the like. Representative alkali oralkaline earth metal salts include sodium, lithium, potassium, calcium,magnesium, and the like. Further pharmaceutically acceptable saltsinclude, when appropriate, nontoxic ammonium, quaternary ammonium, andamine cations formed by direct reaction with the drug carboxylic acid orby using counterions such as halide, hydroxide, carboxylate, sulfate,phosphate, nitrate, sulfonate and aryl sulfonate.

“Pharmaceutically acceptable carrier” or “pharmaceutically acceptableexcipient” includes any and all solvents, dispersion media, coatings,antibacterial and antifungal agents, isotonic and absorption delayingagents and the like. The use of such media and agents forpharmaceutically active substances is well known in the art. Exceptinsofar as any conventional media or agent is incompatible with theactive ingredient, its use in the therapeutic compositions of theinvention is contemplated. Supplementary active ingredients can also beincorporated into the compositions.

“Prodrug” is meant to indicate a compound that may be converted underphysiological conditions or by solvolysis to a biologically activecompound described herein. Thus, the term “prodrug” refers to aprecursor of a biologically active compound that is pharmaceuticallyacceptable. A prodrug may be inactive when administered to a subject,i.e. an ester, but is converted in vivo to an active compound, forexample, by hydrolysis to the free carboxylic acid. The prodrug compoundoften offers advantages of solubility, tissue compatibility or delayedrelease in a mammalian organism (see, e.g., Bundgard, H., Design ofProdrugs (1985), pp. 7-9, 21-24 (Elsevier, Amsterdam). A discussion ofprodrugs is provided in Higuchi, T., et al., “Pro-drugs as NovelDelivery Systems,” A.C.S. Symposium Series, Vol. 14, and inBioreversible Carriers in Drug Design, ed. Edward B. Roche, AmericanPharmaceutical Association and Pergamon Press, 1987, both of which areincorporated in full by reference herein. The term “prodrug” is alsomeant to include any covalently bonded carriers, which release theactive compound in vivo when such prodrug is administered to a mammaliansubject. Prodrugs of an active compound, as described herein, may beprepared by modifying functional groups present in the active compoundin such a way that the modifications are cleaved, either in routinemanipulation or in vivo, to the parent active compound. Prodrugs includecompounds wherein a hydroxy, amino or mercapto group is bonded to anygroup that, when the prodrug of the active compound is administered to amammalian subject, cleaves to form a free hydroxy, free amino or freemercapto group, respectively. Examples of prodrugs include, but are notlimited to, acetate, formate and benzoate derivatives of an alcohol oracetamide, formamide and benzamide derivatives of an amine functionalgroup in the active compound and the like.

“Subject” refers to an animal, such as a mammal, for example a human.The methods described herein can be useful in both human therapeuticsand veterinary applications. In some embodiments, the patient is amammal, and in some embodiments, the patient is human.

The term “in vivo” refers to an event that takes place in a subject'sbody.

The term “in vitro” refers to an event that takes places outside of asubject's body. For example, an in vitro assay encompasses any assay runoutside of a subject assay. In vitro assays encompass cell-based assaysin which cells alive or dead are employed. In vitro assays alsoencompass a cell-free assay in which no intact cells are employed.

Unless otherwise stated, structures depicted herein are also meant toinclude compounds which differ only in the presence of one or moreisotopically enriched atoms. For example, compounds having structureswherein a hydrogen is replaced by a deuterium or tritium, or a carbon isreplaced by ¹³C- or ¹⁴C-enriched carbon are within the scope of thisinvention.

The compounds of the present invention may also contain unnaturalproportions of atomic isotopes at one or more of atoms that constitutesuch compounds. For example, the compounds may be radiolabeled withradioactive isotopes, such as for example tritium (H), iodine-125 (¹²⁵I)or carbon-14 (¹⁴C). All isotopic variations of the compounds of thepresent invention, whether radioactive or not, are encompassed withinthe scope of the present invention.

When ranges are used herein for physical properties, such as molecularweight, or chemical properties, such as chemical formulae, allcombinations and subcombinations of ranges and specific embodimentstherein are intended to be included. The term “about” when referring toa number or a numerical range means that the number or numerical rangereferred to is an approximation within experimental variability (orwithin statistical experimental error), and thus the number or numericalrange may vary from, for example, between 1% and 15% of the statednumber or numerical range. The term “comprising” (and related terms suchas “comprise” or “comprises” or “having” or “including”) includes thoseembodiments, for example, an embodiment of any composition of matter,composition, method, or process, or the like, that “consist of” or“consist essentially of” the described features.

Abbreviations used herein have their conventional meaning within thechemical and biological arts.

Human blood contains white blood cells (leukocytes) which are furtherclassified as neutrophils, lymphocytes (with B- and T-subtypes),monocytes, eosinophils, and basophils. Several of these classes ofleukocytes, neutrophils, eosinophils, basophils and lymphocytes, areinvolved in inflammatory disorders. LFA-1, also known as CD11a/CD18 orthe integrin αLβ2, is one of a group of leukointegrins which areexpressed on most leukocytes, and is considered to be the lymphoidintegrin which interacts with a number of ICAMs as ligands. Theexpression of LFA-1 in humans is almost exclusively limited to cells ofthe immune system, particularly the extracellular surface of leukocytes,including lymphocytes. As such, LFA-1 regulates the adhesion, migration,proliferation, and inflammatory response of lymphocytes, particularlyT-lymphocytes (T-cells), in normal immune function as well as in anumber of inflammatory and autoimmune disease settings. DisruptingLFA-1/ICAM interactions, and thus the immune/inflammatory response,provides for reduction of inflammation.

For example, ICAM-1 (CD54) is a member of the ICAM family of adhesionreceptors (ICAM-1, ICAM-2, ICAM-3, ICAM-4) in the immunoglobulin proteinsuper family, and is expressed on activated leukocytes, dermalfibroblasts, and endothelial cells. It is normally expressed on theendothelial cells lining the vasculature, and is upregulated uponexposure to cytokines or other inflammatory signals such as IL-1, LPSand TNF during immune/inflammatory initiation.

Functional studies of the binding of LFA-1 and ICAM-1 have shown thatthis interaction is crucial in processes which contribute to diseasemechanism including: leukocyte and lymphocyte adhesion to vascularendothelial cells; their extravasation from the vasculature at a site ofinflammation; homotypic interactions between lymphocytes, as well asinteractions between T-cells and dendritic cells in inflamed tissue; theformation of the immunologic synapse and in the transmission ofcostimulatory signals in concert with MHC/T-cell Receptor crucial forlymphocyte proliferation and cytokine release. Consequently, antagonistsof LFA-1/ICAM binding may provide blockade of the adhesive, migratory,proliferative, and inflammatory signaling components of lymphocytemediated inflammation. As such, they may provide more comprehensiveinhibition of T-cell mediated diseases than is currently offered bysteroid or calcincurin antagonist (e.g., cyclosporinc) immunomodulators.Without being bound by mechanistic theory, antagonists of LFA-1 may showanti-inflammatory and immunosuppressive effects on lymphocytes in vitroand in T-cell mediated diseases in vivo, particularly when administeredsystemically to treat inflammatory diseases throughout the body, or whenadministered locally to treat local manifestations of inflammatorydiseases.

The compound of Formula I:

has been found to be an effective inhibitor of LFA-1 interactions withICAM-1. It is a member of a class of directly competitive inhibitors ofLFA-1, binding to ICAM's binding site on LFA-1 directly, and thusexcludes ICAM binding. Directly competitive inhibitors of LFA-1 mayoffer the potential for more effective modulation of the inflammatoryand/or immunologic response than allosteric inhibitors provide becausethese inhibitors occlude the binding site more effectively.

Additionally, the compound of Formula I has a rapid systemic clearancerate. LFA-1 interaction with ICAMs exerts various systemic effectsthroughout the body. Treatment of a disorder using an LFA-1 antagonistmay result in unwanted effects due to LFA-1 antagonist activity inunwanted locations, for example, other than at the site ofadministration. The present invention utilizes the compound of Formula Iwhich is cleared quickly from systemic circulation. The compound ofFormula I may have minimal systemic LFA-1 antagonist activity. In someembodiments, the compound of Formula I may have undetectable systemicLFA-1 antagonist activity. Therefore, the compound of Formula I may beparticularly well suited for treatment of a disorder mediated by theinteraction between LFA-1 and ICAM-1, where localized treatment isdesirable and/or where such localized treatment is administered for manymonths or years.

The systemic clearance rate can be calculated by various means known inthe art. For example, the clearance rate for a drug may be calculatedfrom an analysis of the drug concentration time profile for the drugconcentration time profile for the rate of disappearance of a drug fromthe plasma following administration of the formulation, for exampleafter a single intravenous injection. One of skill in the art could usea variety of methods to calculate and determine systemic clearancerates. For example, the rate of disappearance may be measured byanalysis of the absorption, distribution, metabolism and excretion of aradiolabelled form of a drug or other means of measuring the level ofdrug in plasma, such as gas chromatography (Sapirstein et al., 1955, Am.Jour. Physiol., Vol. 181, pp. 330; U.S. Pat. No. 4,908,202), liquidchromatography-mass spectrometry methods (LCMS) or HPLC methods. Asanother example, the clearance rate may be calculated by introducing theformulation to the subject by continuous intravenous infusion until anequilibrium is reached at which the plasma level of the substance (asdetermined by analysis of plasma samples) is steady, at which point theinfusion rate is equal to the rate of clearance from plasma (Earle etal., 1946, Proc. Soc. Exp. Biol. Med., Vol. 62, pp. 262 ff.)

Rapid systemic clearance may be through clearance or metabolism in theliver, kidney or other organs. Where clearance occurs in a particularorgan, the clearance rate is related to the blood flow to thatparticular organ. By knowing the mechanism in which a compound iscleared for a particular species, the clearance rate for other animalsmay be calculated by allometric scaling. The compound of the presentinvention is known to be cleared through the liver in rats. Based on therate of clearance calculated in rat, the clearance of the compound maybe scaled for various animals based on the known blood flow in ratscompared to other animals (see Davies and Morris, “PhysiologicalParameters in Laboratory Animals and Humans” Pharmaceutical Research(1993) 10:1093-5). For example, 100% of rat hepatic blood flow would beapproximately 55 mL/min/kg while 100% of human hepatic blood flow wouldbe approximately 20 mL/min/kg. The compound of Formula I has a clearancerate in rats of greater than 100 mL/min/kg.

In order to develop clinically useful therapeutics, drug candidates needto be chemically pure enough to administer to a subject and of anacceptable physical form in order to be formulated in pharmaceuticallyacceptable dosage forms. One advantageous route to obtain higher purity,reproducibility of physical form, and stability is to identify one ormore useful crystalline forms. The capacity to exist in differentcrystalline forms is known as polymorphism and is known to occur in manyorganic molecules. These different crystalline forms are known as“polymorphic modifications” or “polymorphs.” While polymorphicmodifications have the same chemical composition, they differ inpacking, geometric arrangement, and other descriptive properties of thecrystalline solid state. As such, these modifications may have differentsolid-state physical properties to affect, for example, the solubility,dissolution rate, bioavailability, chemical and physical stability,flowability, fractability, and compressibility of the compound as wellas the safety and efficacy of drug products based on the compound. Inthe process of preparing a polymorph, further purification, in terms ofgross physical purity or optical purity, may be accomplished as well.

A number of different forms, including crystalline forms, of thecompound of Formula I have been discovered. While crystallization isoften performed on organic compounds, it is not predictable in advanceas to which conditions will provide suitable conditions to lead toformation of a particular crystalline form. Further, it is notpredictable as to which particular crystalline form will provide thenecessary mixture of physical properties, nonlimiting examples of whichare described above, to yield a desirable drug dosage form, onceformulated.

Experimental Instrumentation and Conditions

Fourier Transform-Raman Spectroscopy (FT-Raman) was performed with aBruker RFS100 instrument, using Nd:YAG 1064 nm excitation, 300 mW laserpower, Ge detector, using 64 scans over the range of 25-3500 cm⁻¹, andwith 2 cm⁻¹ resolution.

Power X-ray Diffraction (PXDR) was performed with a Bruker D8 AdvanceX-ray diffractometer with CuKα-radiation. The standard measuringconditions were: tube power 35 kV/45 mA; step size 0.017° (20); steptime 105±5 sec; scanning range 2°-50° (20); divergence slit equal tovariable V12; the samples were rotated; a Vantecl detector was used; theopening angle 3°; channel number 360±10; the y-axis shows the valueintensity/number of active detector channels/sec; silicon single crystalsample holders; and the sample dimensions depth/diameter was 0.1 mm/˜12mm.

Thermogravimetric-Fourier transform Infrared Spectroscopy (TG-FTIR) wasperformed with a Netzsch Thermo-Microbalance TG 209 coupled with aBruker FT-IR Spectrometer Vector 22, using an aluminum crucible (open orwith a microhole), under a nitrogen atmosphere, and at a heating rate of10° C./min over the range of 25° C. to 350° C.

Differential Scanning Calorimetry (DSC) was performed with a PerkinElmer Differential Scanning Calorimeter 7, using closed gold crucibles,a heating rate of 10° C. min⁻¹ or 20° C. min⁻¹ over a range from −50° C.to 250° C.

Dynamic Vapor Sorption (DVS) analysis was performed with a SurfaceMeasurement Systems DVS-1 water vapor sorption analyzer. The experimentswere run by placing the sample on a quartz holder on top of amicrobalance, and allowing the sample to equilibrate at 50% relativehumidity (r.h.) before starting the pre-defined humidity program. Theprogram proceeded in the following steps: 1 hour at 50% r.h.; 50% to 0%r.h. at a rate of 5% r.h. change per hour; 5 hours at 0% r.h; 0% r.h to96% r.h. at 5% r.h change per hour; 5 hours at 95% r.h.; 95% r.h. to 50%r.h. at a rate of 5% r.h. change per hour, and followed by one hour at50% r.h.

Amorphous Form

The reaction product, resulting from synthesis of the compound ofFormula I using the synthetic methods described in U.S. Pat. No.7,314,938, was isolated by concentration from an ethyl acetate extract.It may be further purified to remove calcium impurities or convert thecalcium salt to the free acid. The free acid thus obtained has aFT-Raman chromatogram with broad peaks (FIG. 1), and an X-ray powderdiffraction chromatogram with no peaks (FIG. 2), thus revealing that thereaction product is an amorphous form. The amorphous form has athermogravimetric/Fourier transform infrared chromatogram whichdemonstrates that the amorphous form loses about 5.1% of its mass astemperature is raised to just below 190° C., which is attributed to lossof ethyl acetate. As the temperature is raised above 190° C., theamorphous form begins to decompose, as shown in FIG. 3. The purity ofthe amorphous solid as determined by high pressure liquid chromatographyis 96.6% of the total area under the curve as observed at 220 nm, and97.1% of the total area under the curve at 254.4 nm (Table 1).

Crystallization in selected solvents and under selected conditionsresulted in the discovery of several crystalline forms with distinctphysical behavior.

Crystalline Form A

The amorphous form (free acid, 129 mg) was suspended in 0.2 mlacetonitrile which had been previously dried over molecular sieves andlet stand for 3 days. Another addition of 0.5 ml dry acetonitrile wasmade and the suspension was exposed to ultrasonic radiation. Thesuspension was then stirred for one day, filtered and rinsed with 1.5 mldry acetonitrile to produce crystalline Form A. The crystalline Form Awas also produced by slurrying amorphous form in methyl ethyl ketone(MEK). As a third alternative, slurrying the amorphous form in waterwill also provide crystalline Form A.

The crystalline Form A was characterized by several analyticaltechniques. High pressure liquid chromatography was performed on anAgilent HP1100 instrument, with analysis by Agilent Chemstationsoftware, using a YMC ODS AQ, 3 μm, 350×4.6 mm ID column, and 0.1%o-phosphoric acid in water as Eluent A and 0.1% o-phosphoric acid inacetonitrile as Eluent B. The chromatography was performed with a flowrate of 1 ml/min, with a gradient program consisting of 35% B at T=0, to75% B at 25 min, to 90% B at 30 min, return to 35% B at 30.1 min, andcontinue to 35 min at 35% B. Alternatively, a Zorbax SB C18 50×4.6 mm IDcolumn was used, Eluent C was 0.1% TFA in water and Eluent D was 0.1%TFA in acetonitrile, with a ratio of C:D equal to 2:8, with a flow rateof 1 ml/min. The column temperature was controlled at 40° C. Detectionwas performed at either 220 nm or 254 nm.

Form A has a high pressure liquid chromatogram comprising a total areaunder the curve of 98.0% for Form A, as observed at 220 nm, and a totalarea under the curve of 98.5% for Form A when observed at 254 nm (Table1A). The method of producing crystalline Form A removes more impuritiesthat were introduced to the compound of Formula I throughout thesynthetic route, which may be due to one or more of by-products of thesynthesis, impurities introduced with reagents used in the synthesis,trapped organic solvents used in prior reaction steps, such as methylenechloride, and contaminants due to degradation of an intermediate productof the synthetic sequence. Removal of methylene chloride is particularlyimportant in preparation of formulations for ocular application.Additionally, the level of methylene chloride must be lower than thatpermitted by the FDA guidelines. Impurities which must be removed fromthe product of the synthetic process include residual palladium catalystand residual organic solvents including MEK, ethyl acetate, THF, andtoluene. Other impurities which are intermediates and/or startingmaterials of the synthesis include compounds 12, 18, and 19 (shown inScheme 5), which may be designated byproducts of the synthesis.

TABLE 1A HPLC Purity of Amorphous Form and Crystalline Form A. Form ofthe compound Area % Area % of Formula I (220 nm) (254 nm) Amorphous Form96.6 97.1 Crystalline Form A 98.0 98.5

Form A was also analyzed by chiral high pressure liquid chromatographyto determine the chiral purity of the S enantiomer. The mobile phase was350 ml acetonitrile mixed with 650 ml of water which had previously beenadjusted to pH 3.0 with formic acid. The chromatography was isocratic,using a Chiralcel OJ-RH column, 150×4.6 mm, temperature controlled to40° C., and detection was performed at 260 nm.

TABLE 1B Stereochemical Purity of Crystalline Form A. Form of thecompound of Formula I Stereochemical Purity Crystalline Form A 98.5% Senantiomer

Crystalline Form A was analyzed by ¹H-Nuclear Magnetic Resonancespectroscopy. The resultant spectrum showed a number of the peaks whichwere narrowed in comparison to that of the amorphous form, and removalof trapped ethyl acetate was accomplished.

Crystalline Form A, when analyzed by Raman spectroscopy, has achromatogram which has narrowed peaks relative to the above amorphousform, as is shown in the overlaid spectra of both forms in FIG. 4.Several peaks were shifted relative to the amorphous form. The Ramanchromatogram of crystalline Form A is shown in FIG. 5. Crystalline FormA comprises a powder x-ray diffraction pattern as shown in FIG. 6. Thepeak assignments corresponding to the diffraction pattern forcrystalline Form A and their relative intensities are listed in Table 2.

TABLE 2 Peak Assignments and Intensities of the PXDR Pattern forCrystalline Form A. Angle d value Intensity Intensity % 2-Theta °Angstrom Cps % 5.67 15.6 7 15 7.55 11.7 11 22 8.79 10.0 6 11 9.70 9.1 2244 10.09 8.8 17 34 11.41 7.7 7 14 14.82 6.0 6 11 17.47 5.07 23 47 17.665.02 18 36 18.15 4.88 39 78 18.83 4.71 26 51 19.37 4.58 14 28 20.00 4.4431 62 20.22 4.39 24 47 20.60 4.31 14 28 21.07 4.21 17 33 21.42 4.14 50100 22.68 3.92 39 78 22.91 3.88 22 45 23.18 3.84 15 30 23.52 3.78 15 2924.60 3.62 29 58 24.77 3.59 26 51 25.40 3.50 22 45 26.19 3.40 17 3426.73 3.33 17 33 26.89 3.31 19 38 27.65 3.22 11 21 28.72 3.11 24 4929.74 3.00 10 20 30.45 2.93 9 17 31.81 2.81 11 21 34.79 2.58 12 25

Thermogravimetric analysis graph of crystalline Form A is shown in FIG.7. A mass loss of about 2.5% between room temperature and 180° C. isobserved. From the coupled FTIR analysis carried out simultaneously, theloss is attributable to loss of water. Without being bound by theory,the results suggest that Form A is a hydrate, with a theoretical massloss of 2.8%, and that the water of hydration may be derived from theamorphous form or may have been introduced adventitiously in thecrystallization experiment.

The graph of differential scanning calorimetry of crystalline Form A,performed at a rate of change of 10° C./min, is shown in FIG. 8. Adouble peak is seen at about 145° C., and correlates with the loss ofwater observed in the TG-FTIR. Additionally, crystalline Form A wasanalyzed using DVS. FIG. 9 and FIG. 10 show its behavior over thestandard experimental protocol FIG. 9 shows the change in mass ofcrystalline Form A over the timecourse of the experiment, firstdecreasing in mass as the relative humidity is decreased, stabilizing atthe lowest relative humidity of 0.4%, then increasing with increasingrelative humidity, and stabilizing at 95% relative humidity. The amountof mass loss is consistent with the presence of a hemi hydrate (0.5 molewater per mole free acid) and the mass gain at 95% relative humidity isconsistent with the presence of 1.1 mole water per mole free acid. Thematerial at the start of the experiment has a water content of about 0.9mole water per mole free acid. The behavior of crystalline Form A versusthe relative humidity axis of this experiment is shown in FIG. 10. Asecond TG-FTIR analysis was performed on crystalline Form A materialafter it had been treated according to the DVS experimental conditions.This data is shown in FIG. 11 and demonstrates that the changes in massseen over the course of the DVS analysis are reversible as the samecurve is obtained.

Crystalline Form B

Crystalline Form B was produced from crystalline Form A, by suspending100 mg of crystalline Form A in ethyl acetate, which had been previouslydried over molecular sieves, stirring for four days, and then filteringto isolate crystalline Form B. It was also formed to an extent byslurrying Form A in tert-butyl methyl ether. Crystalline Form B wasanalyzed by most of the same techniques as crystalline Form A. TheFT-Raman spectrum of Crystalline Form B is shown in FIG. 12. The PXDRpattern of crystalline Form B is distinct from that of crystalline FormA, and is shown in FIG. 13. The peak assignments and their relativeintensities are listed in Table 3.

TABLE 3 Peak Assignments and Intensities for the PXDR Pattern ofCrystalline Form B. Angle d value Intensity Intensity % 2-Theta °Angstrom Cps % 7.70 11.5 7 21 10.50 8.4 7 23 11.36 7.8 6 20 12.14 7.3 2474 13.61 6.5 11 34 14.27 6.2 9 28 15.50 5.71 12 37 15.90 5.57 8 25 17.095.19 33 100 18.53 4.79 30 91 19.20 4.62 17 53 19.51 4.55 29 89 20.754.28 15 45 21.17 4.19 13 41 21.60 4.11 15 44 22.48 3.95 17 51 23.21 3.8329 90 23.82 3.73 18 55 24.79 3.59 13 40 25.57 3.48 12 37 26.68 3.34 1235 29.68 3.01 9 28

TG-FTIR data for Form B is displayed in FIG. 14. Residual ethyl acetateand water, in the amount of 0.87% of the total mass, was released fromForm B as the temperature was increased to 240° C., and may beattributed to solvent adsorbed to the surface of Form B. DVS analysis ofForm B is shown in FIG. 15 and FIG. 16, mass change versus time in FIG.15 and mass change versus relative humidity in FIG. 16, respectively.The mass loss upon reduction of the relative humidity to 0.4% is 0.30%and 0.19% mass gain is seen upon equilibration at 95% relative humidity.The decomposition of Form B itself occurred at the same temperature asthat observed for Form A. The graph of the DSC analysis of Form B isshown in FIG. 17, where the peak at 209° C. may be attributed to meltingof Form B. Accordingly, this form does not appear to be hygroscopic andappears to be consistent with an anhydrate which does not convert to ahydrate over the time scale of the DVS experiment (50 hours, and 5 hoursat 95% relative humidity).

Crystalline Form C

Crystalline Form C was produced by suspending 104 mg of crystalline FormA in ethanol, which had been dried over molecular sieves, and stirringthe resultant slurry for four days. Crystalline Form C was isolated uponfiltering. The FT-Raman chromatogram of crystalline Form C is shown inFIG. 18. The PXDR graph of Crystalline Form C (FIG. 19) shows that adistinct and new crystalline form has been isolated. The peakassignments and relative intensities for PXDR pattern for crystallineForm C are listed in Table 4. The graph of the TG-FTIR data forcrystalline Form C is shown in FIG. 20. As the temperature is raised,the first mass loss is attributable to water and ethanol loss, up toabout 120° C. The pronounced loss at about 155° C. of about 3.86% isattributable to loss of ethanol, and may be consistent with crystallineForm C existing as a hemi ethanol solvate, which converts to Form A upondrying in a humid atmosphere.

TABLE 4 Peak Assignments and Intensities for the PXDR Pattern of Form C.Angle d value Intensity Intensity % 2-Theta ° Angstrom Cps % 4.80 18.430 88 5.27 16.8 5 15 6.56 13.5 8 23 8.37 10.6 10 30 9.61 9.2 12 36 10.738.2 15 44 11.41 7.7 5 16 12.37 7.1 8 24 16.40 5.40 12 36 16.92 5.24 1132 17.51 5.06 14 42 17.80 4.98 33 98 18.80 4.72 11 33 19.40 4.57 10 3019.99 4.44 26 77 20.38 4.35 31 92 20.68 4.29 20 59 20.91 4.25 14 4121.52 4.13 34 100 22.08 4.02 19 57 22.65 3.92 28 83 23.48 3.79 27 8023.95 3.71 21 62 24.29 3.66 21 60 24.58 3.62 22 63 25.33 3.51 19 5525.70 3.46 28 83 27.76 3.21 10 29 28.63 3.12 13 38 29.04 3.07 14 4229.42 3.03 14 42 29.68 3.01 12 35 33.21 2.70 7 20 34.00 2.64 9 27 34.532.60 10 29

Crystalline Form D

Crystalline Form D was produced by suspending 97 mg of Form A in 1.0 mlof water, stirring for four days, and then filtering to isolatecrystalline Form D. The FT-Raman chromatogram is shown in FIG. 21. ThePXDR pattern of crystalline Form D is shown in FIG. 22, and representsanother novel crystalline form. The peak assignments and intensities forthe PXDR pattern are listed in Table 5.

TABLE 5 Peak Assignments and Intensities for the PXDR Pattern ofCrystalline Form D. Angle d value Intensity Intensity % 2-Theta °Angstrom Cps % 5.14 17.2 34 52 6.13 14.4 5 8 7.42 11.9 25 38 8.30 10.634 53 8.86 10.0 21 33 10.15 8.7 16 25 10.36 8.5 13 20 12.87 6.9 14 2213.06 6.8 6 10 17.63 5.03 62 95 17.83 4.97 27 41 18.03 4.92 44 68 18.624.76 29 44 18.83 4.71 30 46 19.10 4.64 19 29 19.45 4.56 19 29 19.63 4.5217 27 20.09 4.42 18 29 20.87 4.25 9 15 21.18 4.19 27 42 21.37 4.16 23 3621.72 4.09 65 100 22.20 4.00 15 24 22.30 3.98 15 23 22.52 3.95 15 2422.95 3.87 32 49 23.30 3.82 35 54 23.67 3.76 16 25 24.35 3.65 12 1924.80 3.59 60 93 24.93 3.57 33 52 25.18 3.53 32 50 25.52 3.49 15 2325.87 3.44 14 21 26.34 3.38 17 26 26.54 3.36 31 48 28.01 3.18 24 3728.19 3.16 18 28 28.53 3.13 49 76 28.84 3.09 10 15 29.21 3.06 13 2129.75 3.00 10 15 30.42 2.94 8 12 30.81 2.90 11 17 31.38 2.85 9 14 31.562.83 15 23 32.47 2.76 8 12 32.81 2.73 12 18 33.25 2.69 8 12 33.52 2.67 711 33.88 2.64 9 14 34.31 2.61 9 14 34.40 2.61 9 14 34.63 2.59 7 11TG-FTIR data is shown in FIG. 23 and is consistent with conversion ofcrystalline Form D into crystalline Form A upon heating. A mass loss ofabout 23.5% was observed about 110° C., but does not appear to beconsistent with a dehydration step converting a hydrate to an anhydrate,but appears to be consistent with the loss of water from the surface ofthe solid.

Crystalline Form E

Crystalline Form E was produced similarly to that of crystalline Form A,using 103 mg of the Amorphous Form described above, suspending theamorphous Form in 0.7 ml acetonitrile, previously dried over molecularsieves, and seeded with crystalline Form A. After stirring, crystallineForm E was isolated by filtration, and investigated while still in wetstage. Crystalline Form E converts into crystalline Form A upon drying.The FT-Raman chromatogram of crystalline Form E is shown in FIG. 24, andits PXDR pattern is shown in FIG. 25. The assignment of peaks and theirintensities for the PXDR of crystalline Form E is listed in Table 6. Thedata from the TG-FTIR analysis of Form E is shown in FIG. 26. The largeloss of mass of 14.15% which stabilizes prior to reaching 190° C., isprobably due to residual water and acetonitrile in the wet sample. Astep attributable to dehydration of a solvate is not consistent with thedata in FIG. 26.

TABLE 6 Peak Assignments and Intensities for the PXDR Pattern of Form E.Angle d value Intensity Intensity % 2-Theta ° Angstrom Cps % 5.12 17.321 87 6.13 14.4 4 15 7.40 11.9 13 54 8.26 10.7 22 90 8.82 10.0 14 5710.14 8.7 8 34 10.30 8.6 8 32 12.81 6.9 10 40 17.59 5.04 24 100 18.124.89 9 35 18.60 4.77 10 40 19.03 4.66 10 40 20.19 4.40 6 24 21.10 4.2113 54 21.78 4.08 12 51 22.44 3.96 8 33 22.87 3.89 14 57 23.33 3.81 10 3924.92 3.57 15 63 25.09 3.55 18 73 26.53 3.36 7 29 28.06 3.18 9 36 28.543.13 9 39 29.65 3.01 5 22 30.78 2.90 5 20 31.50 2.84 5 21 32.74 2.73 623

The above methods can be used to manufacture crystalline Forms A, B, C,D, and E. While the manufacture of each of crystalline Forms A, B, C, D,and E has been exemplified employing a specific solvent system andcrystallization conditions, it is envisioned that each form can beobtained employing a different solvent or combination of solvents and/ordifferent crystallization conditions. Further, crystals of any ofcrystalline Forms A, B, C, D, or E may be added to solutions or slurriesof the amorphous form to seed the crystallization of that specific form.Therefore the above description is not meant to limit the invention inany way.

Additionally, it is envisioned that any of the crystalline Forms A, B,C, D, and/or E may be treated to regenerate an amorphous form. In someembodiments of the invention, while the use of the higher level ofpurity of Forms A, 3B, C, D, and/or E may be desirable, it is alsodesirable to utilize an amorphous form in the formulation therein todeliver therapeutically effective amounts of the compound of Formula I.An amorphous form with equivalent purity to that of crystalline Form A,B, C, D, or E was produced by dissolving the crystalline form in aqueoussodium bicarbonate, acidifying, and extracting the compound of Formula Iinto ethyl acetate. Removal of the ethyl acetate yielded the amorphousform having the same purity as that of any of crystalline Forms A, B, C,D, or/and E.

A partial list of useful solvents includes, i.e., for example, water,aliphatic solvents such as pentane, petroleum ether, and hexane;aromatic solvents such as toluene and xylene, aliphatic ketones andesters such as methyl ethyl ketone, acetone ethyl acetate, and butylacetate, alcohols, such as ethyl alcohol, propyl alcohol, and methylalcohol, acetonitrile, ethers, such as ethyl ether, tert-butyl methylether (TBME), and tetrahydrofuran, alkenes and alkynes, alkenyl estersand alcohols, alkynyl esters and alcohols, and aromatic esters andalcohols.

In some of the embodiments of the invention, the purity of the amorphousform of the compound of Formula I as measured by high pressure liquidchromatography is greater than about 90%, about 90.5%, about 91.0%,about 91.5%, about 92.0%, about 92.5%, about 93.0%, about 93.5%, about94.0%, about 94.5%, about 95.0%, about 95.5%, about 96.0%, about 96.5%,about 97.0%, about 97.5%, about 98.0%, about 98.5%, about 99.0%, about99.5%, or about 99.9% total area under the curve as observed at about220 nm. In some embodiments of the invention, the amorphous form of thecompound of Formula I is about 100.0% pure as measured by high pressureliquid chromatography as area under the curve as observed at about 220nm.

In some of the embodiments of the invention, the purity of the amorphousform of the compound of Formula I as measured by high pressure liquidchromatography is greater than about 90%, about 90.5%, about 91.0%,about 91.5%, about 92.0%, about 92.5%, about 93.0%, about 93.5%, about94.0%, about 94.5%, about 95.0%, about 95.5%, about 96.0%, about 96.5%,about 97.0%, about 97.5%, about 98.0%, about 98.5%, about 99.0%, about99.5%, or about 99.9% total area under the curve as observed at about254 nm or about 260 nm. In some embodiments of the invention, theamorphous form of the compound of Formula I is about 100.0% pure asmeasured by high pressure liquid chromatography as area under the curveas observed at about 254 nm or about 260 nm.

In some of the embodiments of the invention, the purity of crystallineForm A of the compound of Formula I as measured by high pressure liquidchromatography is greater than about 90%, about 90.5%, about 91.0%,about 91.5%, about 92.0%, about 92.5%, about 93.0%, about 93.5%, about94.0%, about 94.5%, about 95.0%, about 95.5%, about 96.0%, about 96.5%,about 97.0%, about 97.5%, about 98.0%, about 98.5%, about 99.0%, about99.5%, or about 99.9% total area under the curve as observed at about220 nm. In some embodiments of the invention, the crystalline Form A ofthe compound of Formula I is about 100.0% pure as measured by highpressure liquid chromatography as area under the curve as observed atabout 220 nm.

In some of the embodiments of the invention, the purity of crystallineForm A of the compound of Formula I as measured by high pressure liquidchromatography is greater than about 90%, about 90.5%, about 91.0%,about 91.5%, about 92.0%, about 92.5%, about 93.0%, about 93.5%, about94.0%, about 94.5%, about 95.0%, about 95.5%, about 96.0%, about 96.5%,about 97.0%, about 97.5%, about 98.0%, about 98.5%, about 99.0%, about99.5%, or about 99.9% total area under the curve as observed at about254 nm or about 260 nm. In some embodiments of the invention, thecrystalline Form A of the compound of Formula I is about 99.9% pure asmeasured by high pressure liquid chromatography as area under the curveas observed at about 254 nm or about 260 nm.

In some of the embodiments of the invention, the purity of crystallineForm B of the compound of Formula I as measured by high pressure liquidchromatography is greater than about 90%, about 90.5%, about 91.0%,about 91.5%, about 92.0%, about 92.5%, about 93.0%, about 93.5%, about94.0%, about 94.5%, about 95.0%, about 95.5%, about 96.0%, about 96.5%,about 97.0%, about 97.5%, about 98.0%, about 98.5%, about 99.0%, about99.5%, or about 99.9% total area under the curve as observed at about220 nm. In some embodiments of the invention, the crystalline Form B ofthe compound of Formula I is about 100.0% pure as measured by highpressure liquid chromatography as area under the curve as observed atabout 220 nm.

In some of the embodiments of the invention, the purity of crystallineForm B of the compound of Formula I as measured by high pressure liquidchromatography is greater than about 90%, about 90.5%, about 91.0%,about 91.5%, about 92.0%, about 92.5%, about 93.0%, about 93.5%, about94.0%, about 94.5%, about 95.0%, about 95.5%, about 96.0%, about 96.5%,about 97.0%, about 97.5%, about 98.0%, about 98.5%, about 99.0%, about99.5%, or about 99.9% total area under the curve as observed at about254 nm or about 260 nm. In some embodiments of the invention, thecrystalline Form B of the compound of Formula I is about 100.0% pure asmeasured by high pressure liquid chromatography as area under the curveas observed at about 254 nm or about 260 nm.

In some of the embodiments of the invention, the purity of crystallineForm C of the compound of Formula I as measured by high pressure liquidchromatography is greater than about 90%, about 90.5%, about 91.0%,about 91.5%, about 92.0%, about 92.5%, about 93.0%, about 93.5%, about94.0%, about 94.5%, about 95.0%, about 95.5%, about 96.0%, about 96.5%,about 97.0%, about 97.5%, about 98.0%, about 98.5%, about 99.0%, about99.5%, or about 99.9% total area under the curve as observed at about220 nm. In some embodiments of the invention, the crystalline Form C ofthe compound of Formula I is about 100.0% pure as measured by highpressure liquid chromatography as area under the curve as observed atabout 220 nm.

In some of the embodiments of the invention, the purity of crystallineForm C of the compound of Formula I as measured by high pressure liquidchromatography is greater than about 90%, about 90.5%, about 91.0%,about 91.5%, about 92.0%, about 92.5%, about 93.0%, about 93.5%, about94.0%, about 94.5%, about 95.0%, about 95.5%, about 96.0%, about 96.5%,about 97.0%, about 97.5%, about 98.0%, about 98.5%, about 99.0%, about99.5%, or about 99.9% total area under the curve as observed at about254 nm or about 260 nm. In some embodiments of the invention, thecrystalline Form C of the compound of Formula I is about 100.0% pure asmeasured by high pressure liquid chromatography as area under the curveas observed at about 254 nm or about 260 nm.

In some of the embodiments of the invention, the purity of crystallineForm D of the compound of Formula I as measured by high pressure liquidchromatography is greater than about 90%, about 90.5%, about 91.0%,about 91.5%, about 92.0%, about 92.5%, about 93.0%, about 93.5%, about94.0%, about 94.5%, about 95.0%, about 95.5%, about 96.0%, about 96.5%,about 97.0%, about 97.5%, about 98.0%, about 98.5%, about 99.0%, about99.5%, or about 99.9% total area under the curve as observed at about220 nm. In some embodiments of the invention, the crystalline Form D ofthe compound of Formula I is about 100.0% pure as measured by highpressure liquid chromatography as area under the curve as observed atabout 220 nm.

In some of the embodiments of the invention, the purity of crystallineForm D of the compound of Formula I as measured by high pressure liquidchromatography is greater than about 90%, about 90.5%, about 91.0%,about 91.5%, about 92.0%, about 92.5%, about 93.0%, about 93.5%, about94.0%, about 94.5%, about 95.0%, about 95.5%, about 96.0%, about 96.5%,about 97.0%, about 97.5%, about 98.0%, about 98.5%, about 99.0%, about99.5%, or about 99.9% total area under the curve as observed at about254 nm or about 260 nm. In some embodiments of the invention, thecrystalline Form D of the compound of Formula I is about 100.0% pure asmeasured by high pressure liquid chromatography as area under the curveas observed at about 254 nm or about 260 nm.

In some of the embodiments of the invention, the purity of crystallineForm E of the compound of Formula I as measured by high pressure liquidchromatography is greater than about 90%, about 90.5%, about 91.0%,about 91.5%, about 92.0%, about 92.5%, about 93.0%, about 93.5%, about94.0%, about 94.5%, about 95.0%, about 95.5%, about 96.0%, about 96.5%,about 97.0%, about 97.5%, about 98.0%, about 98.5%, about 99.0%, about99.5%, or about 99.9% total area under the curve as observed at about220 nm. In some embodiments of the invention, the crystalline Form E ofthe compound of Formula I is about 100.0% pure as measured by highpressure liquid chromatography as area under the curve as observed atabout 220 nm.

In some of the embodiments of the invention, the purity of crystallineForm E of the compound of Formula I as measured by high pressure liquidchromatography is greater than about 90%, about 90.5%, about 91.0%,about 91.5%, about 92.0%, about 92.5%, about 93.0%, about 93.5%, about94.0%, about 94.5%, about 95.0%, about 95.5%, about 96.0%, about 96.5%,about 97.0%, about 97.5%, about 98.0%, about 98.5%, about 99.0%, about99.5%, or about 99.9% total area under the curve as observed at about254 nm or about 260 nm. In some embodiments of the invention, thecrystalline Form E of the compound of Formula I is about 100.0% pure asmeasured by high pressure liquid chromatography as area under the curveas observed at about 254 nm or about 260 nm.

In some of the embodiments of the methods of manufacture of theinvention, the chiral purity of the amorphous form of the compound ofFormula I as measured by chiral chromatography at 260 nm is greater thanabout 75%, about 75.5%, about 76%, about 76.5%, about 77%, about 77.5%,about 78%, about 78.5%, about 79%, about 79.5%, about 80%, about 80.5%,about 81%, about 81.5%, about 82%, about 82.5%, about 83%, about 83.5%,about 84%, about 84.5%, about 85%, about 85.5%, about 86%, about 86.5%,about 87%, about 87.5%, about 88%, about 88.5%, about 89%, about 89.5%,about 90%, about 90.5%, about 91.0%, about 91.5%, about 92.0%, about92.5%, about 93.0%, about 93.5%, about 94.0%, about 94.5%, about 95.0%,about 95.5%, about 96.0%, about 96.5%, about 97.0%, about 97.5%, about98.0%, about 98.5%, about 99.0%, about 99.5%, or about 99.9% of theS-enantiomer. In some embodiments, the chiral purity of the amorphousform of the compound of Formula I as measured by chiral chromatographyat 260 nm is about 100%.

In some of the embodiments of the methods of manufacture of theinvention, the chiral purity of crystalline Form A of the compound ofFormula I as measured by chiral chromatography at 260 nm is greater thanabout 75%, about 75.5%, about 76%, about 76.5%, about 77%, about 77.5%,about 78%, about 78.5%, about 79%, about 79.5%, about 80%, about 80.5%,about 81%, about 81.5%, about 82%, about 82.5%, about 83%, about 83.5%,about 84%, about 84.5%, about 85%, about 85.5%, about 86%, about 86.5%,about 87%, about 87.5%, about 88%, about 88.5%, about 89%, about 89.5%,about 90%, about 90.5%, about 91.0%, about 91.5%, about 92.0%, about92.5%, about 93.0%, about 93.5%, about 94.0%, about 94.5%, about 95.0%,about 95.5%, about 96.0%, about 96.5%, about 97.0%, about 97.5%, about98.0%, about 98.5%, about 99.0%, about 99.5%, or about 99.9% of theS-enantiomer. In some embodiments, the chiral purity of the Form A ofthe compound of Formula I as measured by chiral chromatography at 260 nmis about 100%.

In some of the embodiments of the methods of manufacture of theinvention, the chiral purity of crystalline Form B of the compound ofFormula I as measured by chiral chromatography at 260 nm is greater thanabout 75%, about 75.5%, about 76%, about 76.5%, about 77%, about 77.5%,about 78%, about 78.5%, about 79%, about 79.5%, about 80%, about 80.5%,about 81%, about 81.5%, about 82%, about 82.5%, about 83%, about 83.5%,about 84%, about 84.5%, about 85%, about 85.5%, about 86%, about 86.5%,about 87%, about 87.5%, about 88%, about 88.5%, about 89%, about 89.5%,about 90%, about 90.5%, about 91.0%, about 91.5%, about 92.0%, about92.5%, about 93.0%, about 93.5%, about 94.0%, about 94.5%, about 95.0%,about 95.5%, about 96.0%, about 96.5%, about 97.0%, about 97.5%, about98.0%, about 98.5%, about 99.0%, about 99.5%, or about 99.9% of theS-enantiomer. In some embodiments, the chiral purity of the Form B ofthe compound of Formula I as measured by chiral chromatography at 260 nmis about 100%.

In some of the embodiments of the methods of manufacture of theinvention, the chiral purity of crystalline Form C of the compound ofFormula I as measured by chiral chromatography at 260 nm is greater thanabout 75%, about 75.5%, about 76%, about 76.5%, about 77%, about 77.5%,about 78%, about 78.5%, about 79%, about 79.5%, about 80%, about 80.5%,about 81%, about 81.5%, about 82%, about 82.5%, about 83%, about 83.5%,about 84%, about 84.5%, about 85%, about 85.5%, about 86%, about 86.5%,about 87%, about 87.5%, about 88%, about 88.5%, about 89%, about 89.5%,about 90%, about 90.5%, about 91.0%, about 91.5%, about 92.0%, about92.5%, about 93.0%, about 93.5%, about 94.0%, about 94.5%, about 95.0%,about 95.5%, about 96.0%, about 96.5%, about 97.0%, about 97.5%, about98.0%, about 98.5%, about 99.0%, about 99.5%, or about 99.9% of theS-enantiomer. In some embodiments, the chiral purity of the Form C ofthe compound of Formula I as measured by chiral chromatography at 260 nmis about 100%.

In some of the embodiments of the methods of manufacture of theinvention, the chiral purity of crystalline Form D of the compound ofFormula I as measured by chiral chromatography at 260 nm is greater thanabout 75%, about 75.5%, about 76%, about 76.5%, about 77%, about 77.5%,about 78%, about 78.5%, about 79%, about 79.5%, about 80%, about 80.5%,about 81%, about 81.5%, about 82%, about 82.5%, about 83%, about 83.5%,about 84%, about 84.5%, about 85%, about 85.5%, about 86%, about 86.5%,about 87%, about 87.5%, about 88%, about 88.5%, about 89%, about 89.5%,about 90%, about 90.5%, about 91.0%, about 91.5%, about 92.0%, about92.5%, about 93.0%, about 93.5%, about 94.0%, about 94.5%, about 95.0%,about 95.5%, about 96.0%, about 96.5%, about 97.0%, about 97.5%, about98.0%, about 98.5%, about 99.0%, about 99.5%, or about 99.9% of theS-enantiomer. In some embodiments, the chiral purity of the Form D ofthe compound of Formula I as measured by chiral chromatography at 260 nmis about 100%.

In some of the embodiments of the methods of manufacture of theinvention, the chiral purity of crystalline Form E of the compound ofFormula I as measured by chiral chromatography at 260 nm is greater thanabout 75%, about 75.5%, about 76%, about 76.5%, about 77%, about 77.5%,about 78%, about 78.5%, about 79%, about 79.5%, about 80%, about 80.5%,about 81%, about 81.5%, about 82%, about 82.5%, about 83%, about 83.5%,about 84%, about 84.5%, about 85%, about 85.5%, about 86%, about 86.5%,about 87%, about 87.5%, about 88%, about 88.5%, about 89%, about 89.5%,about 90%, about 90.5%, about 91.0%, about 91.5%, about 92.0%, about92.5%, about 93.0%, about 93.5%, about 94.0%, about 94.5%, about 95.0%,about 95.5%, about 96.0%, about 96.5%, about 97.0%, about 97.5%, about98.0%, about 98.5%, about 99.0%, about 99.5%, or about 99.9% of theS-enantiomer. In some embodiments, the chiral purity of the Form E ofthe compound of Formula I as measured by chiral chromatography at 260 nmis about 100%.

In some of the embodiments of the methods of manufacture of theinvention, the amorphous form of the compound of Formula I has less thanabout 2.0%, about 1.9%, about 1.8%, about 1.7%, about 1.6%, about 1.5%,about 1.4%, about 1.3%, about 1.2%, about 1.1%, about 1.0%, about 0.9%,about 0.8%, about 0.7%, about 0.6%, about 0.5%, about 0.4%, about 0.3%,about 0.2%, about 0.1%, about 0.09%, about 0.08%, about 0.07%, about0.06%, about 0.05%, about 0.04%, about 0.03%, about 0.02%, about 0.01%,or about 0.009% of any one impurity introduced, obtained or produced asa result of the chemical synthesis, as measured by chromatography atabout 220 nm. In some embodiments, the impurity is a by-product of thesynthesis.

In some of the embodiments of the methods of manufacture of theinvention, crystalline Form A of the compound of Formula I has less thanabout 2.0%, about 1.9%, about 1.8%, about 1.7%, about 1.6%, about 1.5%,about 1.4%, about 1.3%, about 1.2%, about 1.1%, about 1.0%, about 0.9%,about 0.8%, about 0.7%, about 0.6%, about 0.5%, about 0.4%, about 0.3%,about 0.2%, about 0.1%, about 0.09%, about 0.08%, about 0.07%, about0.06%, about 0.05%, about 0.04%, about 0.03%, about 0.02%, about 0.01%,or about 0.009% of any one impurity introduced, obtained or produced asa result of the chemical synthesis, as measured by chromatography at 220nm. In some embodiments, the impurity is a by-product of the synthesis.

In some of the embodiments of the methods of manufacture of theinvention, crystalline Form B of the compound of Formula I has less thanabout 2.0%, about 1.9%, about 1.8%, about 1.7%, about 1.6%, about 1.5%,about 1.4%, about 1.3%, about 1.2%, about 1.1%, about 1.0%, about 0.9%,about 0.8%, about 0.7%, about 0.6%, about 0.5%, about 0.4%, about 0.3%,about 0.2%, about 0.1%, about 0.09%, about 0.08%, about 0.07%, about0.06%, about 0.05%, about 0.04%, about 0.03%, about 0.02%, about 0.01%,or about 0.009% of any one impurity introduced, obtained or produced asa result of the chemical synthesis, as measured by chromatography atabout 220 nm. In some embodiments, the impurity is a by-product of thesynthesis.

In some of the embodiments of the methods of manufacture of theinvention, crystalline Form C of the compound of Formula I has less thanabout 2.0%, about 1.9%, about 1.8%, about 1.7%, about 1.6%, about 1.5%,about 1.4%, about 1.3%, about 1.2%, about 1.1%, about 1.0%, about 0.9%,about 0.8%, about 0.7%, about 0.6%, about 0.5%, about 0.4%, about 0.3%,about 0.2%, about 0.1%, about 0.09%, about 0.08%, about 0.07%, about0.06%, about 0.05%, about 0.04%, about 0.03%, about 0.02%, about 0.01%,or about 0.009% of any one impurity introduced, obtained or produced asa result of the chemical synthesis, as measured by chromatography at 220nm. In some embodiments, the impurity is a by-product of the synthesis.

In some of the embodiments of the methods of manufacture of theinvention, crystalline Form D of the compound of Formula I has less thanabout 2.0%, about 1.9%, about 1.8%, about 1.7%, about 1.6%, about 1.5%,about 1.4%, about 1.3%, about 1.2%, about 1.1%, about 1.0%, about 0.9%,about 0.8%, about 0.7%, about 0.6%, about 0.5%, about 0.4%, about 0.3%,about 0.2%, about 0.1%, about 1.0%, about 0.9%, about 0.8%, about 0.7%,about 0.6%, about 0.5%, about 0.4%, about 0.3%, about 0.2%, about 0.1%,about 0.09%, about 0.08%, about 0.07%, about 0.06%, about 0.05%, about0.04%, about 0.03%, about 0.02%, about 0.01%, or about 0.009% of any oneimpurity introduced, obtained or produced as a result of the chemicalsynthesis, as measured by chromatography at 220 nm. In some embodiments,the impurity is a by-product of the synthesis.

In some of the embodiments of the methods of manufacture of theinvention, crystalline Form E of the compound of Formula I has less thanabout 2.0%, about 1.9%, about 1.8%, about 1.7%, about 1.6%, about 1.5%,about 1.4%, about 1.3%, about 1.2%, about 1.1%, about 1.0%, about 0.9%,about 0.8%, about 0.7%, about 0.6%, about 0.5%, about 0.4%, about 0.3%,about 0.2%, about 0.1%, about 0.09%, about 0.08%, about 0.07%, about0.06%, about 0.05%, about 0.04%, about 0.03%, about 0.02%, about 0.01%,or about 0.009% of any one impurity introduced, obtained or produced asa result of the chemical synthesis, as measured by chromatography at 220nm. In some embodiments, the impurity is a by-product of the synthesis.

In some of the embodiments of the method of manufacture of theinvention, the amorphous form of the compound of Formula I comprisesless than about 3.0%, about 2.8%, about 2.6%, about 2.4%, about 2.2%,about 2.1%, about 2.0%, about 1.9%, about 1.8%, about 1.7%, about 1.6%,about 1.5%, about 1.4%, about 1.3%, about 1.2%, about 1.1%, about 1.0%,about 0.9%, about 0.8%, about 0.7%, about 0.6%, about 0.5%, about 0.4%,about 0.3%, about 0.2%, about 0.1%, or about 0.09% of total impuritiesintroduced, obtained or produced as a results of the chemical synthesis,as measured by chromatography at 220 nm. In some embodiments theimpurities comprise a by-product of the chemical synthesis.

In some of the embodiments of the method of manufacture of theinvention, crystalline Form A of the compound of Formula I comprisesless than about 3.0%, about 2.8%, about 2.6%, about 2.4%, about 2.2%,about 2.1%, about 2.0%, about 1.9%, about 1.8%, about 1.7%, about 1.6%,about 1.5%, about 1.4%, about 1.3%, about 1.2%, about 1.1%, about 1.0%,about 0.9%, about 0.8%, about 0.7%, about 0.6%, about 0.5%, about 0.4%,about 0.3%, about 0.2%, about 0.1%, or about 0.09% of total impuritiesintroduced, obtained or produced as a results of the chemical synthesis,as measured by chromatography at 220 nm. In some embodiments theimpurities comprise a by-product of the chemical synthesis.

In some of the embodiments of the method of manufacture of theinvention, crystalline Form B of the compound of Formula I comprisesless than about 3.0%, about 2.8%, about 2.6%, about 2.4%, about 2.2%,about 2.1%, about 2.0%, about 1.9%, about 1.8%, about 1.7%, about 1.6%,about 1.5%, about 1.4%, about 1.3%, about 1.2%, about 1.1%, about 1.0%,about 0.9%, about 0.8%, about 0.7%, about 0.6%, about 0.5%, about 0.4%,about 0.3%, about 0.2%, about 0.1%, or about 0.09% of total impuritiesintroduced, obtained or produced as a results of the chemical synthesis,as measured by chromatography at 220 nm. In some embodiments theimpurities comprise a by-product of the chemical synthesis.

In some of the embodiments of the method of manufacture of theinvention, crystalline Form C of the compound of Formula I comprisesless than about 3.0%, about 2.8%, about 2.6%, about 2.4%, about 2.2%,about 2.1%, about 2.0%, about 1.9%, about 1.8%, about 1.7%, about 1.6%,about 1.5%, about 1.4%, about 1.3%, about 1.2%, about 1.1%, about 1.0%,about 0.9%, about 0.8%, about 0.7%, about 0.6%, about 0.5%, about 0.4%,about 0.3%, about 0.2%, about 0.1%, or about 0.09% of total impuritiesintroduced, obtained or produced as a results of the chemical synthesis,as measured by chromatography at 220 nm. In some embodiments theimpurities comprise a by-product of the chemical synthesis.

In some of the embodiments of the method of manufacture of theinvention, crystalline Form D of the compound of Formula I comprisesless than about 3.0%, about 2.8%, about 2.6%, about 2.4%, about 2.2%,about 2.1%, about 2.0%, about 1.9%, about 1.8%, about 1.7%, about 1.6%,about 1.5%, about 1.4%, about 1.3%, about 1.2%, about 1.1%, about 1.0%,about 0.9%, about 0.8%, about 0.7%, about 0.6%, about 0.5%, about 0.4%,about 0.3%, about 0.2%, about 0.1%, or about 0.09% of total impuritiesintroduced, obtained or produced as a results of the chemical synthesis,as measured by chromatography at 220 nm. In some embodiments theimpurities comprise a by-product of the chemical synthesis.

In some of the embodiments of the method of manufacture of theinvention, crystalline Form E of the compound of Formula I comprisesless than about 3.0%, about 2.8%, about 2.6%, about 2.4%, about 2.2%,about 2.1%, about 2.0%, about 1.9%, about 1.8%, about 1.7%, about 1.6%,about 1.5%, about 1.4%, about 1.3%, about 1.2%, about 1.1%, about 1.0%,about 0.9%, about 0.8%, about 0.7%, about 0.6%, about 0.5%, about 0.4%,about 0.3%, about 0.2%, about 0.1%, or about 0.09% of total impuritiesintroduced, obtained or produced as a results of the chemical synthesis,as measured by chromatography at 220 nm. In some embodiments theimpurities comprise a by-product of the chemical synthesis.

Methods of Manufacture of the Compound of Formula I

In one embodiment, the compound of Formula I was synthesized as in thefollowing Schemes 1-7. Alternate steps were used in the process asdescribed below. The variants of this overall route yield superioryields, cost of goods and superior chiral purity compared to previouslydescribed methods. The final product of this synthesis yieldscrystalline Form A directly.

A first alternative protecting strategy produces compound 5, a tritylprotected species as shown in Scheme 1. The synthesis begins byreductively aminating 3, 5, dichlorobenzaldehyde, compound 1, with1-chloro-2-aminoethane and sodium cyanoborohydride in 35% yield.Cyclization of compound 2 using aluminum chloride catalysis and ammoniumchloride at 185° C. provided compound 3 in 91% yield. Protection of thefree amine of compound 3 as the trityl protected species affordedcompound 4 in 89% yield. A carboxylic acid functionality was introducedby treatment of compound 4 with n-butyllithium (nBuLi) andTetramethylethylenediamine (TMEDA), with subsequent introduction ofcarbon dioxide, to produce compound 5 in 75% yield.

Bromophenylalanine was used as the starting material for the right handportion of the final molecule as shown in Scheme 2. t-Butylcarbamate(Boc) protection of the amino group was accomplished, using sodiumbicarbonate (3 equivalents), t-butyl dicarbonate (Boc₂O, 1.1 equivalent)in dioxane and water, to obtain compound 7 in 98% yield. A methylsulfone functionality was introduced by treating the bromo compound 7with copper iodide (0.4 equivalents), cesium carbonate (0.5equivalents), L-proline (0.8 equivalents), and the sodium salt ofmethanesulfinic acid (3.9 equivalents) in dimethylsulfoxide (DMSO) at95-100° C. for a total of 9 hours, with two further additions of copperiodide (0.2 equivalents) and L-proline (0.4 equivalents) during thatperiod. Compound 8 was isolated in 96% yield. The carboxylic acid ofcompound 8 was converted to the benzyl ester, compound 9, in 99% yield,using benzyl alcohol (1.1 equivalent), dimethylaminopyridine (DMAP, 0.1equivalent) and N-(3-dimethylaminopropyl)-N-ethylcarbodiimide (EDC, 1.0equivalent). The amino group of compound 9 is deprotected by adding a 4Nsolution of HCl in dioxane to compound 9 at 0° C. in methylene chloride.The HCl salt of the free amino species, compound 10 was isolated in 94%yield.

Compound 5 was treated with triethylamine (TEA, 5 equivalents) and2-(7-Aza-1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate (HATU, 1.25 equivalents) for 10 minutes indimethylformamide (DMF), and then compound 10 was added to the solution.After stirring at room temperature for 18 hours, the product, compound11 was isolated in 70% yield. Removal of the trityl protecting group wasaccomplished by treating compound 1, with HCl in dioxane (4N, excess) atroom temperature for 2 hours, diethyl ether added, and the solidproduct, compound 12, was isolated by filtration in 95% yield.

The benzofuranyl carbonyl moiety of the compound of Formula I wasprepared using two alternative schemes, Scheme 4 and Scheme 4″. In oneembodiment, the benzofuranyl carbonyl moiety was prepared by protectingthe hydroxyl group of compound 13 by reacting withtert-butyldimethylsilyl chloride (1.0 equivalents) and triethylamine(TEA, 1.1 equivalents) in acetone, to give compound 14 in 79% yield. Asolution of compound 14 in methanol was then treated with sodiumborohydride (1.0 equivalent) at room temperature overnight. The reactionwas quenched with an addition of acetone, stirred at room temperaturefor a further 2.5 hours, aqueous HCl (4N) was added with the temperaturecontrolled to below 28 C, tetrahydrofuran (THF) was added, and thesolution stirred overnight under argon and in the absence of light. Theproduct, compound 15, was isolated quantitatively by extraction intomethylene chloride, concentrated at low heat, and used without furtherpurification. The triflate ester, compound 16, was produced in 69% yieldfrom compound 15 by reacting it withN-phenyl-bis(trifluoromethanesulfonimide) (1.0 equivalent) in methylenechloride for 72 hours. Compound 16 in a mixture of DMF, methanol, andtriethylamine, was added to a prepared solution of palladium acetate,diphenyl, DMF and methanol in an autoclave. Carbon monoxide was chargedinto the autoclave to a pressure of 8 bar, and the reaction mixture washeated at 70° C. for 6 hours. After workup, compound 17 was isolated in91% yield. Lithium hydroxide (4 equivalents) in methanol and water wasused to hydrolyze the ester and permit the isolation of compound 18 in97% yield.

In one embodiment, the benzofuranyl carbonyl moiety of the compound ofFormula I was prepared according to Scheme 4″. By way of an Arbuzovreaction, diethyl 2-(1,3-dioxolan-2-yl)ethylphosphonate, compound 1″,was prepared from 2-(2-bromoethyl)-1,3-dioxolane by the addition oftriethyl phosphate. After removal of ethyl bromide through distillationat 210° C. the crude reaction mixture was cooled and then by way ofvacuum distillation, compound 1″ was collected as a colorless oil in 94%yield.

In the next step, n-butyllithium (2.15 equivalents) in hexane was cooledto −70° C. and diisopropylamine (2.25 equivalents) was added whilekeeping the temperature below −60° C. Compound 1″ (1 equivalent)dissolved in tetrahydrofuran (THF) was added over 30 min at −70° C.After 10 min, diethyl carbonate (1.05 equivalents) dissolved in THF wasadded over 30 min keeping the reaction temperature below −60° C. Afterstirring for one hour at −60° C., the reaction was allowed to warm to15° C. and furan-2-carbaldehyde (1.3 equivalents) dissolved in THF wasadded. After stirring for 20 hrs at room temperature, the reaction wasrotary evaporated to dryness to yield ethyl2-((1,3-dioxolan2-yl)methyl-3-(furan-2-yl)acrylate, compound 5″. Crudecompound 5″ was used directly in the next reaction.

The crude compound 5″ (1 equivalent) was dissolved in ethanol and addedto a mixture of water and phosphoric acid (85%, 15 equivalents) over 30min while keeping the temperature below 50° C. After stirring for 20 hrsat room temperature, another 200 ml of phosphoric acid (85%) was addedand the mixture was heated to 50° C. for an additional two hrs. Afterremoval of ethanol by rotary evaporation, the material was extractedwith toluene, washed with water, dried with sodium sulfate, treated withcharcoal, filtered and dried down to an oil. This oil was distilled toafford ethyl benzofuran-6-carboxylate, compound 6″, (bp 111-114.5° C.)which crystallized on standing. Compound 6″ was recovered at 57% yieldbased on compound 1″.

Compound 6″ (875 mmol) was dissolved in methanol and tetrahydrofuran(THF). Sodium hydroxide (4 M, 3 equivalents) was added and the reactionwas stirred overnight. After concentration via rotary evaporation, theaqueous solution was extracted with methyl tert-butyl ether (MTBE),acidified to pH 2 with the addition of hydrochloric acid (HCl) andcooled resulting in fine crystals of benzofuran-6-carboxylic acid, i.e.,compound 18. Compound 18 was isolated, washed with water and dried to afinal yield of 97% yield.

The benzofuran carboxylic acid 18 was treated with oxalyl chloride (1.2equivalents) and a catalytic amount of DMF, stirring for 5.5 hours untila clear solution was obtained. The solvent was removed under reducedpressure and the acid chloride of compound 18 was stored under argonuntil use, on the next day. The acid chloride, in methylene chloride wasadded slowly to a methylene chloride solution of the compound of FormulaI and diisopropylethylamine (DIPEA) which was cooled to 0-5° C. Thereaction was not permitted to rise above 5° C., and after completion ofaddition, was stirred at 5° C. for a further 0.5 hour. Upon aqueousworkup and extraction with methylene chloride, the product, compound 19,was isolated in quantitative yield.

In one embodiment, the benzyl ester of compound 19 was removed bytransfer hydrogenolysis using 10% palladium on carbon, using formic acidand triethylamine in a 5:1 mixture of methanol:THF, to produce thecompound of Formula I in 95% yield. An alternate strategy to convertcompound 19 to Formula I is provided in Scheme 7 below.

A final step of slurrying in methyl ethylketone (MEK) produced Form A ofthe compound of Formula I. The product was washed with water to removeresidual MEK. Alternatively, the product of the hydrogenolysis step wasslurried in acetonitrile yielded Form A of the compound of Formula I.

Taking the compound of Formula I directly as the crude reaction productafter transfer hydrogenolysis, and reconcentrating down from a solutionin methylene chloride, the amorphous form of the compound of Formula Iwas obtained in 97% purity.

An alternative protection strategy in this synthetic approach isillustrated in Scheme 6.

In this alternative approach, Boc-protection was used for the ringnitrogen in the intermediates 20, 21, and 22. Compound 5 was deprotectedwith HCl in dioxane to produce compound 20 in better than 97% yield.Boc-protection was introduced, using di-tert-butyl dicarbonate (1.1equivalent), and compound 21 was obtained in better than 95% yield.Compound 10 was coupled with compound 21 to obtain compound 22, usingHATU and triethylamine in DMF. The product, compound 22, was obtained inquantitative yield, and greater than 90% purity. Deprotection with HClyields the compound of Formula I in 97.4% yield and the synthesisrejoins the process described in Scheme 5.

One of the advantages of the foregoing synthesis is the use of a benzylester to protect the carboxylic acid originating in thebromophenylalanine starting material. The resultant transferhydrogenolysis of compound 19 produces the compound of Formula I with amuch higher optical purity (98.5% S enantiomer) than the optical purityobtained (79-94.5% S enantiomer) by hydrolysis of a corresponding methylester.

An alternate strategy to convert compound 19 into Formula I is providedby base hydrolysis of the benzyl ester of compound 19.

Compound 19 ((S)-benzyl2-(2-(benzofuran-6carbonyl)-5,7-dichloro-1,2,3,4-tetrahydroisoquinoline-6-carboxamido)-3-(3-methylsulfonyl)phenyl)propanoate)(70.9 mmol) was dissolved in dioxane and water was added. This solutionwas cooled to 8° C. Over 45 minutes, NaOH (0.5 M) was added to 68.0mmol. After stirring for 2 hrs, the dioxane was removed by rotaryevaporation. The aqueous solution was extracted twice with toluene toremove unreacted starting material. Ethyl actetate was added to theaqueous layer and with vigorous stirring the aqueous layer was acidifiedto pH 2 with HCl (4 M aqueous). After stirring, the layers wereseparated and the aqueous layer was extracted with ethyl acetate. Thecombined ethyl acetate fractions were washed with brine, dried withsodium sulfate and evaporated to dryness resulting in a foam which was95% pure by HPLC and had a 94.8% ee. This foam was dissolved in methylethylketone (MEK) and was seeded with crystals (99% pure, 99% ee) whichresulted in thick crystallization. After stirring for 24 hr, thesuspension was filtered and washed with water and dried under vacuum.The yield of Formula I was 77% with a purity of 98.9% and 97.9% eeoptical purity. An additional crop of Formula I (>98% pure) was obtainedthrough concentration of the mother liquor.

A general strategy to convert a compound of Formula AA is provided bybase hydrolysis of the ester to yield the compound of Formula I.

The compound of Formula AA may be reacted with a base in a solvent toaccomplish the base-catalyzed saponification of Formula AA to yield thecompound of Formula I.

The saponification solvent may be any industrially available solventsuch as an aprotic solvent, a protic solvent, a polar solvent, anon-polar solvent, an ionic solvent, or a pressurized gas such assupercritical carbon dioxide. In various embodiments, the solvent is anaprotic solvent such as dioxane or tetrahydrofuran. Variously, thesolvent may be selected from hexane, benzene, toluene, 1,4-dioxane,chloroform, diethyl ether, dichloromethane, tetrahydrofuran, ethylacetate, acetone, dimethylformamide, acetonitrile, dimethyl sulfoxide,n-butanol, isopropanol, n-propanol, ethanol, methanol, water, andcombinations thereof. The base may be any base used for saponificationreactions. In various embodiments, the base is a hydroxide such aspotassium hydroxide or sodium hydroxide or lithium hydroxide.

In various embodiments, the R group is any carbon containing moiety.Within the group where R is any carbon containing moiety, R may beselected from lower alkyl, lower alkenyl, lower alkynyl,cyclo(lower)alkyl, cyclo(lower)alkenyl, aryl, aralkyl, heterocyclyl, andheteroaryl, any of which can be substituted or unsubstituted. In variousembodiments, the lower alkyl group is methyl, ethyl, propyl, isopropyl,butyl, pentyl, isobutyl, t-butyl, or hexyl. In the embodiment of FormulaA, the R group is a benzyl group.

A general strategy to convert a compound of Formula AA is provided byacid hydrolysis of the ester to yield the compound of Formula I.

The compound of Formula AA may be reacted with an acid in a solvent toaccomplish the acid-catalyzed hydrolysis of Formula AA to yield thecompound of Formula I.

The hydrolysis solvent may be any industrially available solvent such asan aprotic solvent, a protic solvent, a polar solvent, a non-polarsolvent, an ionic solvent, or a pressurized gas such as supercriticalcarbon dioxide. In various embodiments, the solvent is an aproticsolvent such as dioxane or tetrahydrofuran. Variously, the solvent maybe selected from hexane, benzene, toluene, 1,4-dioxane, chloroform,diethyl ether, dichloromethane, tetrahydrofuran, ethyl acetate, acetone,dimethylformamide, acetonitrile, dimethyl sulfoxide, n-butanol,isopropanol, n-propanol, ethanol, methanol, water, formic acid, aceticacid, trifluoroacetic acid, and combinations thereof. The acid may beany acid used for hydrolysis reactions. In various embodiments, the acidis a mineral acid. In various embodiments, the acid is selected fromhydrogen chloride, sulfuric acid, and phosphoric acid. In variousembodiments, the acid is trifluoroacetic acid.

In various embodiments, the R group is any carbon containing moiety.Within the group where R is any carbon containing moiety, R may beselected from lower alkyl, lower alkenyl, lower alkynyl,cyclo(lower)alkyl, cyclo(lower)alkenyl, aryl, aralkyl, heterocyclyl, andheteroaryl, any of which can be substituted or unsubstituted. In variousembodiments, the lower alkyl group is methyl, ethyl, propyl, isopropyl,butyl, pentyl, isobutyl, t-butyl, or hexyl. In the embodiment of FormulaAA where the compound is Formula A, the R group is a benzyl group. Invarious embodiments, the invention is directed to compounds of FormulaAA wherein R is any carbon containing moiety. In various embodiments ofFormula AA, the carbon-containing moiety does not include a benzylgroup. In various embodiments, the invention is directed to compounds ofFormula AA wherein R is selected from lower alkyl, lower alkenyl, loweralkynyl, cyclo(lower)alkyl, cyclo(lower)alkenyl, aryl, heterocyclyl, andheteroaryl, any of which can be substituted or unsubstituted. Suchcompounds may be useful as synthetic intermediates of compounds ofFormula I, or as prodrugs of Formula I.

In various embodiments, compounds synthesized according to the inventionmay have various advantages, such as ease of purification, reduced cost,reduced number of synthetic steps, higher overall yields, reducedimpurities, differing impurity profiles, and reduced racemization of thechiral center. In one embodiment, the compound synthesized according tothe invention has an enantiomeric excess (ee) selected from greater thanabout 95% ee, about 96%, about 97%, about 98%, about 99%, and about99.9%. In various embodiments, the compound synthesized according to theinvention has reduced levels of chemical catalyst as an impuritycompared to a compound of Formula I made using palladium as a catalystto remove an ester group to yield the carboxylic acid. For example, invarious embodiments, the compound has less than 100 ppm contaminationwith palladium, or less than 50 ppm, or less than 10 ppm, or less than 1ppm contamination with palladium. In various embodiments, the compoundis essentially free of chemical catalyst.

Methods of Use

Diseases and Disorders for which the Amorphous Form or any of theCrystalline Forms a, B, C, D, and E of the Compound of Formula I areUseful.

Not intending to limit the invention by a single mechanism of action,the methods of the present invention involve the inhibition ofinitiation and progression of inflammation related disease by inhibitingthe interaction between LFA-1 and ICAM-1 by administering the amorphousform or any of the crystalline Forms A, B, C, D, or E, or a combinationthereof, of the compound of Formula I. In some embodiments, such methodsprovide anti-inflammatory effects in-vitro and in-vivo, and are usefulin the treatment of inflammation mediated diseases.

In particular, the amorphous form or any of the crystalline Forms A, B,C, D, or E, or a combination thereof, of the compound of Formula I canmodulate inflammation mediated by leukocytes. The amorphous form or anyof the crystalline Forms A, B, C, D, or E, or a combination thereof, ofthe compound of Formula I can be used as a therapeutic agent in anydisorder in which antibodies to LFA-1 are shown to be effective. In oneembodiment of the invention, a subject is administered the amorphousform or any of the crystalline Forms A, B, C, D, or E, or a combinationthereof, of the compound of Formula I to modulate inflammationassociated with ocular inflammation. Another embodiment of the methods,a subject with inflammation associated with dry eye syndrome isadministered the amorphous form or any of the crystalline Forms A, B, C,D, or E, or a combination thereof, of the compound of Formula I.

One embodiment of the invention provides methods for treating a subjectin need thereof for symptoms of dry eye due to allergies, diabetes,lacrimal gland deficiency, lupus, Parkinson's disease, Sjogren'sdisease, rheumatoid arthritis, or rosaceaby administering the amorphousform or any of the crystalline Forms A, B, C, D, or E, or a combinationthereof, of the compound of Formula I. Another embodiment of theinvention provides methods for treating a subject in need thereof forsymptoms of dry eye disorder due to complications arising from LASIKtherapy for vision correction, use of contact lenses, exposure to aridclimates, exposure to air pollution, exposure to windy climates, orexposure due to cigarette smoke by administering the amorphous form orany of the crystalline Forms A, B, C, D, or E, or a combination thereof,of the compound of Formula I.

The invention also provides methods for treating a subject in needthereof for symptoms of dry eye disorder due to keratoconjunctivitissicca, corneal injury, conjunctival fibrosis, age-related dry eye,Stevens-Johnson syndrome, or congenital alachrima, by administering theamorphous form or any of the crystalline Forms A, B, C, D, or E, or acombination thereof, of the compound of Formula I.

In yet other embodiments, methods are providing for treating a subjectin need thereof for symptoms of dry eye disorder due to pharmacologicalside effects of other drugs being taken by the patient by administeringthe amorphous form or any of the crystalline Forms A, B, C, D, or E, ora combination thereof, of the compound of Formula I. Additionally,methods are provided for treating a subject in need thereof for symptomsof dry eye disorder due to infection by administering the amorphous formor any of the crystalline Forms A, B, C, D, or E, or a combinationthereof, of the compound of Formula I in combination with an antibioticor antimicrobial agent. A subject with symptoms of dry eye disorder dueto eye stress, including that due to computer use, may be treated byadministering the amorphous form or any of the crystalline Forms A, B,C, D, or E, or a combination thereof, of the compound of Formula I.

Other embodiments of the invention provide methods of treating a subjectin need thereof for symptoms of dry eye disorder due to Riley-Daysyndrome, glandular and tissue destruction, ocular cicatricalpemphogoid, blepharitis, autoimmune and other immunodeficient disorders,an inability to blink, by administering the amorphous form or any of thecrystalline Forms A, B, C, D, or E, or a combination thereof, of thecompound of Formula I.

In another aspect of the invention, methods are provided for treating asubject in need thereof for symptoms of psoriasis, of bullous skindiseases, dermatitis, inflammatory bowel disease (including but notlimited to Crohn's disease and ulcerative colitis), hidradentitissupperativa, discoid lupus erythrematosus, erythema multiforme,Whipple's disease, or gluten-sensitive enteropathy, by administering theamorphous form or any of the crystalline Forms A, B, C, D, or E, or acombination thereof, of the compound of Formula I.

In other embodiments of the invention, methods are provided for treatinga subject in need thereof for symptoms of adult respiratory distresssyndrome, pulmonary fibrosis, meningitis, uveitis, eczema, encephalitis,or atopic dermatitis, by administering the amorphous form or any of thecrystalline Forms A, B, C, D, or E, or a combination thereof, of thecompound of Formula I.

In yet another aspect of the invention, methods are provided fortreating a subject in need thereof for symptoms of allergic rhinitis,allergic conjunctivitis, rhinitis, food hypersensitivity, asthma,eczema, or skin hypersensitivity reactions by administering theamorphous form or any of the crystalline Forms A, B, C, D, or E, or acombination thereof, of the compound of Formula I. In other embodiments,methods are provided for treating a subject in need thereof for symptomsof atherosclerosis, rheumatoid arthritis, juvenile chronic arthritis,osteoarthritis, oral lichen planus, systemic lupus erythematosus (SLE)by administering the amorphous form or any of the crystalline Forms A,B, C, D, or E, or a combination thereof, of the compound of Formula I.

Methods of the present invention also include methods for treating asubject in need thereof for symptoms of diabetes mellitus, multiplesclerosis, idiopathic demyelinating polyneuropathy, Guillain-Barresyndrome, Reynaud's syndrome, or autoimmune thrombocytopenia, byadministering the amorphous form or any of the crystalline Forms A, B,C, D, or E, or a combination thereof, of the compound of Formula I. Inother embodiments of the invention, methods are provided for treating asubject in need thereof for symptoms of thyroiditis, immune mediatedrenal disease, experimental autoimmune encephalomyelitis, tuberculosis,or sarcoidosis by administering the amorphous form or any of thecrystalline Forms A, B, C, D, or E, or a combination thereof, of thecompound of Formula I. In some other embodiments of the invention,methods are provided for treating a subject in need thereof for symptomsof polymyositis, granulomatosis, vasculitis, pernicious anemia, byadministering the amorphous form or any of the crystalline Forms A, B,C, D, or E, or a combination thereof, of the compound of Formula I.

In another aspect of the invention, methods are provided for treating asubject in need thereof for symptoms of chronic obstructive pulmonarydisease (COPD), eosinophilic pneumonias, idiopathic pulmonary fibrosis,hypersensitivity pneumonitis, bronchitis by administering the amorphousform or any of the crystalline Forms A, B, C, D, or E, or a combinationthereof, of the compound of Formula I.

Additionally, the invention provides methods for treating a subject inneed thereof for symptoms of insulinitis, urticaria, glomerulonephritis,autoimmune chronic active hepatitis, sclerosing cholangitis, diabeticretinopathy, CNS inflammatory disorder, organ injury syndrome secondaryto septicemia or trauma, autoimmune hemolytic anemia, or myastheniagravis by administering the amorphous form or any of the crystallineForms A, B, C, D, or E, or a combination thereof, of the compound ofFormula I. In other embodiments, methods are provided for treating asubject in need thereof for symptoms of antigen-antibody complexmediated diseases, symptoms of malignancies (e.g., B-cell malignanciessuch as chronic lymphocytic leukemia or hairy cell leukemia), nephroticsyndrome, chronic lymphocytic leukemia, or hairy cell leukemia byadministering the amorphous form or any of the crystalline Forms A, B,C, D, or E, or a combination thereof, of the compound of Formula I.

In yet a further aspect of the invention, methods are provided fortreating a subject in need thereof for symptoms of post surgicalinflammation of the eye, graft versus host disease (including but notlimited to corneal, kidney, or islet cell transplantation), host versusgraft disease (including but not limited to corneal, kidney, or isletcell transplantation), or inflammatory response associated withtransplantation, by administering the amorphous form or any of thecrystalline Forms A, B, C, D, or E, or a combination thereof, of thecompound of Formula I. An embodiment of the invention treats a subjectwith symptoms of HIV and rhinovirus infections by administering theamorphous form or any of the crystalline Forms A, B, C, D, or E, or acombination thereof, of the compound of Formula I.

The amorphous form or any of the crystalline Forms A, B, C, D, or E, ora combination thereof, of the compound of Formula I may be formulated ina manner such that a therapeutically effective amount of the compound ofFormula I is delivered only locally, for example, i.e. to the dermis,and a less than therapeutically effective amount of the compound isdistributed systemically. Other formulations of the amorphous form orany of the crystalline Forms A, B, C, D, or E, or a combination thereofof the compound of Formula I, may be chosen such that a therapeuticallyeffective amount of the compound of Formula I is delivered systemically.

Pharmaceutical Compositions

In various embodiments of the methods of the invention, the amorphousform or any of the crystalline Forms A, B, C, D, or E, or a combinationthereof of the compound of Formula I are administered in pharmaceuticalcompositions. The pharmaceutical compositions of the invention comprisepharmaceutically acceptable carriers and excipients as well as theamorphous form or any of the crystalline Forms A, B, C, D, or E, or acombination thereof of the compound of Formula I, in order to formulatethe composition for appropriate administration to the subject.

In some of the embodiments of the invention, the crystalline formremains in crystalline form in the pharmaceutical composition. In otherembodiments, the amorphous form and/or crystalline form is solubilizedand is no longer crystalline. In the latter case, however, the superiorpurity or other physicochemical properties of the amorphous form and/orcrystalline form contributes to, i.e., for example, ease of handling theform of the compound of Formula I to form the composition, superiorstorage capabilities of crystalline form prior to formulation, bettertherapeutic index, tolerability of the compound of Formula I to thesubject, or decreased side effects of the compound of Formula I. Theamorphous form or crystalline Forms A, B, C, D, or E may be milled toprovide desirable properties for formulation.

Administration

Administration of a pharmaceutical composition comprising the amorphousform or any of crystalline Forms A, B, C, D, or E, or a combinationthereof, of the compound of Formula I may be by any suitable means. Insome embodiments, a pharmaceutical composition comprising the amorphousform or any of crystalline Forms A, B, C, D, or E, or a combinationthereof, of the compound of Formula I, is administered by oral,transdermal, by injection, slow release intraocular implantation,aerosol administration.

In another aspect of the invention, a pharmaceutical compositioncomprising the amorphous form or any of crystalline Forms A, B, C, D, orE, or a combination thereof, of the compound of Formula I, isadministered locally to achieve therapeutically effective concentrationlocally and does not distribute systemically at pharmacologicallyeffective concentrations, which may be by oral, transdermal, depot,injection, ocular insert, instillation, inhalation, or pumpadministration. In other embodiments, a pharmaceutical compositioncomprising the amorphous form or any of crystalline Forms A, B, C, D, orE, or a combination thereof, of the compound of Formula I, isadministered locally with a sustained release profile to achievetherapeutically effective concentration locally and does not distributesystemically at pharmacologically effective concentrations, which may beby oral, transdermal, depot, injection, ocular insert, instillation,inhalation, or pump administration. In yet other embodiments, apharmaceutical composition comprising the amorphous form or any ofcrystalline Forms A, B, C, D, or E, or a combination thereof, of thecompound of Formula I, is administered orally, transdermally, viaperiocular implant, or via suppository with a slow release profile.Alternatively, a pharmaceutical composition comprising the amorphousform or any of crystalline Forms A, B, C, D, or E, or a combinationthereof, of the compound of Formula I, is administered topically.

In some embodiments, a pharmaceutical composition comprising theamorphous form or any of crystalline Forms A, B, C, D, or E, or acombination thereof, of the compound of Formula I, have a clearance rateof at least 5% of hepatic blood flow. In humans, this would mean aclearance rate of 1 mL/min/kg. In other embodiments, a pharmaceuticalcomposition comprising the amorphous form or any of crystalline Forms A,B, C, D, or E, or a combination thereof, of the compound of Formula I,have a clearance rate of at least about 10%, 20%, 30%, 40%, 50%, 60%,70%, 80%, 90% or about 100% of hepatic blood flow rate in humans (whichwould be a clearance rate in human liver of 20 mL/min/kg). In yet otherembodiments, a pharmaceutical composition comprising the amorphous formor any of crystalline Forms A, B, C, D, or E, or a combination thereof,of the compound of Formula I, may have a clearance rate of at leastabout 110%, 120%, 130%, 140%, 150%, 175%, 200%, 220%, 240%, 260%, 280%,300%, 320%, 340%, 360%, 380%, 400%, 420%, 440%, 460%, 480%, or 500% ofhepatic blood flow rate in humans.

The clearance rates of a pharmaceutical composition comprising theamorphous form or any of crystalline Forms A, B, C, D, or E, or acombination thereof, of the compound of Formula I, may include clearancerates scaled to humans of approximately 1-500 mL/min/kg. In someembodiments a pharmaceutical composition comprising the amorphous formor any of crystalline Forms A, B, C, D, or E, or a combination thereof,of the compound of Formula I, may have a systemic clearance rate ofapproximately 1 mL/min/kg or greater. In other embodiments, apharmaceutical composition comprising the amorphous form or any ofcrystalline Forms A, B, C, D, or E, or a combination thereof, of thecompound of Formula I, may have a systemic clearance rate ofapproximately 2 mL/min/kg or greater. In other embodiments, apharmaceutical composition comprising the amorphous form or any ofcrystalline Forms A, B, C, D, or E, or a combination thereof, of thecompound of Formula I, may have a systemic clearance rate ofapproximately 3 mL/min/kg or greater. In other embodiments, apharmaceutical composition comprising the amorphous form or any ofcrystalline Forms A, B, C, D, or E, or a combination thereof, of thecompound of Formula I, may have a systemic clearance rate ofapproximately 5 mL/min/kg or greater. In other embodiments, thecompositions of the invention may have a systemic clearance rate ofapproximately 7 mL/min/kg or greater. In some embodiments, apharmaceutical composition comprising the amorphous form or any ofcrystalline Forms A, B, C, D, or E, or a combination thereof, of thecompound of Formula I, may have a systemic clearance rate ofapproximately 10 mL/min/kg or greater. In other embodiments, apharmaceutical composition comprising the amorphous form or any ofcrystalline Forms A, B, C, D, or E, or a combination thereof, of thecompound of Formula I, may have a systemic clearance rate ofapproximately 15 mL/min/kg or greater. In other embodiments apharmaceutical composition comprising the amorphous form or any ofcrystalline Forms A, B, C, D, or E, or a combination thereof, of thecompound of Formula I, may have a systemic clearance rate ofapproximately 20 mL/min/kg or greater. In other embodiments, apharmaceutical composition comprising the amorphous form or any ofcrystalline Forms A, B, C, D, or E, or a combination thereof, of thecompound of Formula I, may have a systemic clearance rate ofapproximately 25 mL/min/kg or greater. In some embodiments, apharmaceutical composition comprising the amorphous form or any ofcrystalline Forms A, B, C, D, or E, or a combination thereof, of thecompound of Formula I, may have a systemic clearance rate ofapproximately 30 mL/min/kg or greater. In some embodiments, apharmaceutical composition comprising the amorphous form or any ofcrystalline Forms A, B, C, D, or E, or a combination thereof, of thecompound of Formula I, may have a systemic clearance rate ofapproximately 40 mL/min/kg or greater. In other embodiments, apharmaceutical composition comprising the amorphous form or any ofcrystalline Forms A, B, C, D, or E, or a combination thereof, of thecompound of Formula I, may have a systemic clearance rate ofapproximately 50 mL/min/kg or greater. In yet other embodiments, apharmaceutical composition comprising the amorphous form or any ofcrystalline Forms A, B, C, D, or E, or a combination thereof, of thecompound of Formula I, may have a systemic clearance rate of at leastabout 60, 65, 70, 75, 80, 85, 90, 95, or 100 mL/min/kg.

If additional therapeutic agents are administered as separatecompositions, they may be administered by the same route or by differentroutes. If additional therapeutic agents are administered in a singlepharmaceutical composition with the amorphous form or any of crystallineForms A, B, C, D, or E, or a combination thereof, of the compound ofFormula I, it may be administered by any suitable route, including butnot limited to oral, transdermal, injection, or topically.

In some embodiments, a pharmaceutical composition comprising theamorphous form or any of crystalline Forms A, B, C, D, or E, or acombination thereof, of the compound of Formula I is administered in asingle dose. A single dose of a pharmaceutical composition comprisingthe amorphous form or any of crystalline Forms A, B, C, D, or E, or acombination thereof, of the compound of Formula I may also be used whenit is co-administered with another substance (e.g., an analgesic) fortreatment of an acute condition.

In some embodiments, a pharmaceutical composition comprising theamorphous form or any of crystalline Forms A, B, C, D, or E, or acombination thereof, of the compound of Formula I is administered inmultiple doses. Dosing may be about once, twice, three times, fourtimes, five times, six times, seven times, eight times, nine times, tentimes or more than ten times per day. Dosing may be about once a year,twice a year, every six months, every 4 months, every 3 months, every 60days, once a month, once every two weeks, once a week, or once everyother day. In another embodiment the administration of thepharmaceutical composition comprising the amorphous form or any ofcrystalline Forms A, B, C, D, or E, or a combination thereof, of thecompound of Formula I, continues for less than about 7 days. In yetanother embodiment the administration continues for more than about 6,10, 14, 28 days, two months, six months, or one year. In some cases,dosing is maintained as long as necessary, e.g., dosing for chronicinflammation.

In another embodiment, a pharmaceutical composition comprising theamorphous form or any of crystalline Forms A, B, C, D, or E, or acombination thereof, of the compound of Formula I, is administered incombination with another therapeutic agent about once per day to about10 times per day. In another embodiment the co-administration of thepharmaceutical composition comprising the amorphous form or any ofcrystalline Forms A, B, C, D, or E, or a combination thereof, of thecompound of Formula I with another therapeutic substance continues forless than about 7 days. In yet another embodiment the co-administrationcontinues for more than about 6, 10, 14, 28 days, two months, sixmonths, or one year. In some cases, co-administered dosing is maintainedas long as necessary, e.g., dosing for chronic inflammation. In someembodiments, the co-administration is in the same composition.

In another embodiment, the co-administration is in separatepharmaceutical compositions. In some embodiments, the co-administrationis concomitant. In some embodiments, the administration of the secondtherapeutic agent is before the administration of the pharmaceuticalcomposition comprising the amorphous form or any of crystalline Forms A,B, C, D, or E, or a combination thereof, of the compound of Formula I.In some embodiments, the administration of the second therapeutic agentis after the administration of the pharmaceutical composition comprisingthe amorphous form or any of crystalline Forms A, B, C, D, or E, or acombination thereof, of the compound of Formula I. In one embodiment,the second therapeutic agent is an analgesic, antibiotic, orantihistamine.

In some embodiments, the amorphous form or any of crystalline Forms A,B, C, D, or E, or a combination thereof, of the compound of Formula I,is present in an amount sufficient to exert a therapeutic effect toreduce symptoms of an immune related disorder by an average of at leastabout 5, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, more than 90%, orsubstantially eliminate symptoms of the immune related disorder. Formany inflammatory diseases, there are well recognized clinicalassessments of therapeutic effect (e.g. PASI score for psoriasis andEASI score for eczema).

In some embodiments, the amorphous form or any of crystalline Forms A,B, C, D, or E, or a combination thereof, of the compound of Formula I,is present in an amount sufficient to decrease neovascularization anderythema in a treated individual by an average of at least about 5, 10,15, 20, 25, 30, 40, 50, 60, 70, 80, 90, more than 90%, or substantiallyeliminate neovascularization.

In some embodiments, the amorphous form or any of crystalline Forms A,B, C, D, or E, or a combination thereof, of the compound of Formula I,is present in an amount sufficient to decrease fibrovascular growth ofan individual by an average of at least about 5, 10, 15, 20, 25, 30, 40,50, 60, 70, 80, 90, more than 90%, or substantially eliminatefibrovascular growth.

Administration of the compositions of the invention may continue as longas necessary. In some embodiments, a composition of the invention isadministered for more than 1, 2, 3, 4, 5, 6, 7, 14, or 28 days. In someembodiments, a composition of the invention is administered for lessthan 28, 14, 7, 6, 5, 4, 3, 2, or 1 day. In some embodiments, acomposition of the invention is administered chronically on an ongoingbasis, e.g., for the treatment of chronic pain.

In some embodiments, the composition of the invention delivers atherapeutically effective amount of the amorphous form or any ofcrystalline Forms A, B, C, D, or E, or a combination thereof, of thecompound of Formula I locally and not systemically. In otherembodiments, the composition of the invention delivers a therapeuticallyeffective amount of the amorphous form or any of crystalline Forms A, B,C, D, or E, or a combination thereof, of the compound of Formula Isystemically.

Dosing for the amorphous form or any of crystalline Forms A, B, C, D, orE, or a combination thereof, of the compound of Formula I, in themethods of the invention may be found by routine experimentation. Thedaily dose can range from about 1×10⁻¹⁰ g to 5000 mg. Daily dose rangemay depend on the form of form of the compound of Formula I e.g., theesters or salts used, and/or route of administration, and/or solubilityof the specific amorphous or crystalline form as described herein. Forexample, for systemic administration, typical daily dose ranges are,e.g. about 1-5000 mg, or about 1-3000 mg, or about 1-2000 mg, or about1-1000 mg, or about 1-500 mg, or about 1-100 mg, or about 10-5000 mg, orabout 10-3000 mg, or about 10-2000 mg, or about 10-1000 mg, or about10-500 mg, or about 10-200 mg, or about 10-100 mg, or about 20-2000 mgor about 20-1500 mg or about 20-1000 mg or about 20-500 mg, or about20-100 mg, or about 50-5000 mg, or about 50-4000 mg, or about 50-3000mg, or about 50-2000 mg, or about 50-1000 mg, or about 50-500 mg, orabout 50-100 mg, about 100-5000 mg, or about 100-4000 mg, or about100-3000 mg, or about 100-2000 mg, or about 100-1000 mg, or about100-500 mg. In some embodiments, the daily dose of the amorphous form orany of crystalline Forms A, B, C, D, or E, or a combination thereof, ofthe compound of Formula I is about 100, 200, 300, 400, 500, 600, 700,800, 900, or 1000 mg. In some embodiments, the daily dose of theamorphous form or any of crystalline Forms A, B, C, D, or E, or acombination thereof, of the compound of Formula I is 0.1 mg. In someembodiments, the daily dose of the amorphous form or any of crystallineForms A, B, C, D, or E, or a combination thereof, of the compound ofFormula I is 1.0 mg. In some embodiments, the daily dose of theamorphous form or any of crystalline Forms A, B, C, D, or E, or acombination thereof, of the compound of Formula I is 10 mg. In someembodiments, the daily dose of the amorphous form or any of crystallineForms A, B, C, D, or E, or a combination thereof, of the compound ofFormula I is 100 mg. In some embodiments, the daily dose of theamorphous form or any of crystalline Forms A, B, C, D, or E, or acombination thereof, of the compound of Formula I is 500 mg. In someembodiments, the daily dose of the amorphous form or any of crystallineForms A, B, C, D, or E, or a combination thereof, of the compound ofFormula I is 1000 mg.

In some embodiments, the amorphous form or any of crystalline Forms A,B, C, D, or E, or a combination thereof, of the compound of Formula I ispresent in an amount sufficient to exert a therapeutic effect to reducesymptoms of a disorder mediated by LFA-1, by an average of at leastabout 5, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, more than 90%, orsubstantially eliminate symptoms of the disorder mediated by LFA-1.

In some embodiments, an effective amount of the amorphous form or any ofcrystalline Forms A, B, C, D, or E, or a combination thereof, of thecompound of Formula I is a daily dose of about 1×10-¹¹, 1×10-¹⁰, 1×10-⁹,1×10⁻⁸, 1×10⁻⁷, 1×10⁻⁶, 1×10⁻⁵, 1×10⁻⁴, 1×10⁻³, 1×10⁻², 1×10⁻¹, 1,1×10¹, 1×10² grams.

For topical delivery to the ocular surface, the typical daily doseranges are, e.g. about 1×10⁻¹⁰ g to 5.0 g, or about 1×10⁻¹⁰ g to 2.5 g,or about 1×10⁻¹⁰ g to 1.00 g, or about 1×10⁻¹⁰ g to 0.5 g, or about1×10⁻¹⁰ g to 0.25 g, or about 1×10⁻¹⁰ g to 0.1 g, or about 1×10⁻¹⁰ g to0.05 g, or about 1×10⁻¹⁰ g to 0.025 g, or about 1×10⁻¹⁰ g to 1×10⁻² g,or about 1×10⁻¹⁰ g to 5×10⁻³ g, or about 1×10⁻¹⁰ g to 2.5×10⁻³ g, orabout 1×10⁻¹⁰ g to 1×10⁻³ g, or about 1×10⁻¹⁰ g to 5×10⁻⁴ g, or 1×10⁻¹⁰g to 2.5×10⁻⁴ g, or about 1×10⁻¹⁰ g to 1×10⁻⁴ g, or about 1×10⁻¹⁰ g to5×10⁻⁵ g, or 1×10⁻¹⁰ g to 2.5×10⁻⁵ g, or about 1×10⁻¹⁰ g to 1×10⁻⁵ g, orabout 1×10⁻¹⁰ g to 5×10⁻⁶ g, or about 1×10⁻⁹ g to 1.00 g, or about1×10⁻⁹ g to 0.5 g, or about 1×10⁻⁹ g to 0.25 g, or about 1×10⁻⁹ g to 0.1g, or about 1×10⁻⁹ g to 0.05 g, or about 1×10⁻⁹ g to 0.025 g, or about1×10⁻⁹ g to 1×10⁻² g, or about 1×10⁻⁹ g to 5×10⁻³ g, or about 1×10⁻⁹ gto 2.5×10⁻³ g, or about 1×10⁻⁹ g to 1×10⁻³ g, or about 1×10⁻⁹ g to5×10⁻⁴ g, or about 1×10⁻⁸ g to 5.0 g, or about 1×10⁻⁸ g to 2.5 g, orabout 1×10⁻⁸ g to 1 g, or about 1×10⁻⁸ g to 0.5 g, or about 1×10⁻⁸ g to0.25 g, or about 1×10⁻⁸ g to 0.1 g, or about 1×10⁻⁸ g to 5×10⁻² g, orabout 1×10⁻⁸ to 5×10⁻² g, or about 1×10⁻⁸ g to 2.5×10⁻² g, or about1×10⁻⁸ g to 1×10⁻² g, or about 1×10⁻⁸ g to 5×10⁻³ g, or about 1×10⁻⁸ to2.5×10⁻³ g, or about 1×10⁻⁸ g to 1×10⁻³ g, or about 1×10⁻⁸ g to 5×10⁻⁴g, or about 1×10⁻⁷ g to 5.0 g, or about 1×10⁻⁷ g to 2.5 g, or about1×10⁻⁷ g to 1 g, or about 1×10⁻⁷ g to 0.5 g, or about 1×10⁻⁷ g to 0.25g, or about 1×10⁻⁷ g to 0.1 g, or about 1×10⁻⁷ g to 5×10⁻² g, or about1×10⁻⁷ to 5×10⁻² g, or about 1×10⁻⁷ g to 2.5×10⁻² g, or about 1×10⁻⁷ gto 1×10⁻² g, or about 1×10⁻⁷ g to 5×10⁻³ g, or about 1×10⁻⁷ to 2.5×10⁻³g, or about 1×10⁻⁷ g to 1×10⁻³ g, or about 1×10⁻⁷ to 5×10⁻⁴ g, or about1×10⁻⁶ g to 5.0 g, or about 1×10⁻⁶ g to 2.5 g, or about 1×10⁻⁶ g to 1 g,or about 1×10⁻⁶ g to 0.5 g, or about 1×10⁻⁶ g to 0.25 g, or about 1×10⁻⁶g to 0.1 g, or about 1×10⁻⁶ g to 5×10⁻² g, or about 1×10⁻⁶ to 5×10⁻² g,or about 1×10⁻⁶ g to 2.5×10⁻² g, or about 1×10⁻⁶ g to 1×10⁻² g, or about1×10⁻⁶ g to 5×10⁻³ g, or about 1×10 to 2.5×10⁻³ g, or about 1×10⁻⁶ g to1×10⁻³ g, or about 1×10⁻⁶ g to 5×10⁻⁴ g, or about 1×10⁻⁵ g to 5 g, orabout 1×10⁻⁵ g to 2.5 g, or about 1×10⁻⁵ g to 1 g, or about 1×10⁻⁵ g to0.5 g, or about 1×10⁻⁵ g to 0.25 g, or about 1×10⁻⁵ g to 0.1 g, or about1×10⁻⁵ g to 0.05 g, or about 1×10⁻⁵ g to 2.5×10⁻² g, or about 1×10⁻⁵ gto 1×10⁻² g, or about 1×10⁻⁵ g to 5×10⁻³ g, or about 1×10⁻⁵ g to2.5×10⁻³ g, or about 1×10⁻⁵ g to 1×10⁻³ g, or about 1×10⁻⁵ g to 5×10⁻⁴g.

In some embodiments, the daily dose of the amorphous form or any ofcrystalline Forms A, B, C, D, or E, or a combination thereof, of thecompound of Formula I is about 1×10⁻¹⁰, about 1×10⁻⁹, about 1×10⁻⁸,about 1×10⁻⁷, about 1×10⁻⁶, about 1×10⁻⁵, about 1×10⁻⁴, about 1×10⁻³ g,about 1×10⁻² g, about 1×10¹ g, or about 1 g. In some embodiments, thedaily dose of the amorphous form or any of crystalline Forms A, B, C, D,or E, or a combination thereof, of the compound of Formula I is about1×10⁻¹⁰ g. In some embodiments, the daily dose of the amorphous form orany of crystalline Forms A, B, C, D, or E, or a combination thereof, ofthe compound of Formula I is about 1×10⁻⁹ g. In some embodiments, thedaily dose of the amorphous form or any of crystalline Forms A, B, C, D,or E, or a combination thereof, of the compound of Formula I is about1×10⁻⁸ g. In some embodiments, the daily dose of the amorphous form orany of crystalline Forms A, B, C, D, or E, or a combination thereof, ofthe compound of Formula I is about 1×10⁻⁷ g. In some embodiments, thedaily dose of the amorphous form or any of crystalline Forms A, B, C, D,or E, or a combination thereof, of the compound of Formula I, is about1×10⁻⁵ g. In some embodiments, the daily dose of the amorphous form orany of crystalline Forms A, B, C, D, or E, or a combination thereof, ofthe compound of Formula I, is about 1×10⁻³ g. In some embodiments, thedaily dose of the amorphous form or any of crystalline Forms A, B, C, D,or E, or a combination thereof, of the compound of Formula I, is about1×10⁻² g. In some embodiments the individual dose ranges from about1×10⁻¹⁰ g to 5.0 g, or about 1×10⁻¹⁰ g to 2.5 g, or about 1×10⁻¹⁰ g to1.00 g, or about 1×10⁻¹⁰ g to 0.5 g, or about 1×10⁻¹⁰ g to 0.25 g, orabout 1×10⁻¹⁰ g to 0.1 g, or about 1×10⁻¹⁰ g to 0.05 g, or about 1×10⁻¹⁰g to 0.025 g, or about 1×10⁻¹⁰ g to 1×10⁻² g, or about 1×10⁻¹⁰ g to5×10⁻³ g, or about 1×10⁻¹⁰ g to 2.5×10⁻³ g, or about 1×10⁻¹⁰ g to 1×10⁻³g, or about 1×10⁻¹⁰ g to 5×10⁻⁴ g, or 1×10⁻¹⁰ g to 2.5×10⁻⁴ g, or about1×10⁻¹⁰ g to 1×10⁻⁴ g, or about 1×10⁻¹⁰ g to 5×10⁻⁵ g, or 1×10⁻¹⁰ g to2.5×10⁻⁵ g, or about 1×10⁻¹⁰ g to 1×10⁻⁵ g, or about 1×10⁻¹⁰ g to 5×10⁻⁶g, or about 1×10⁻⁹ g to 1.00 g, or about 1×10⁻⁹ g to 0.5 g, or about1×10⁻⁹ g to 0.25 g, or about 1×10⁻⁹ g to 0.1 g, or about 1×10⁻⁹ g to0.05 g, or about 1×10⁻⁹ g to 0.025 g, or about 1×10⁻⁹ g to 1×10⁻² g, orabout 1×10⁻⁹ g to 5×10⁻³ g, or about 1×10⁻⁹ g to 2.5×10⁻³ g, or about1×10⁻⁹ g to 1×10⁻³ g, or about 1×10⁻⁹ g to 5×10⁻⁴, or about 1×10⁻⁸ g to5.0 g, or about 1×10⁻⁸ g to 2.5 g, or about 1×10⁻⁸ g to 1 g, or about1×10⁻⁸ g to 0.5 g, or about 1×10⁻⁸ g to 0.25 g, or about 1×10⁻⁸ g to 0.1g, or about 1×10⁻⁸ g to 5×10⁻² g, or about 1×10⁻⁸ to 5×10⁻² g, or about1×10⁻⁸ g to 2.5×10⁻² g, or about 1×10⁻⁸ g to 1×10⁻² g, or about 1×10⁻⁸ gto 5×10⁻³ g, or about 1×10⁻⁸ to 2.5×10⁻³ g, or about 1×10⁻⁸ g to 1×10⁻³g, or about 1×10⁻⁸ g to 5×10 g, or about 1×10⁻⁷ g to 5.0 g, or about1×10⁻⁷ g to 2.5 g, or about 1×10⁻⁷ g to 1 g, or about 1×10⁻⁷ g to 0.5 g,or about 1×10⁻⁷ g to 0.25 g, or about 1×10⁻⁷ g to 0.1 g, or about 1×10⁻⁷g to 5×10⁻² g, or about 1×10⁻⁷ to 5×10⁻² g, or about 1×10⁻⁷ g to2.5×10⁻² g, or about 1×10⁻⁷ g to 1×10⁻² g, or about 1×10⁻⁷ g to 5×10⁻³g, or about 1×10⁻⁷ to 2.5×10⁻³ g, or about 1×10⁻⁷ g to 1×10⁻³ g, orabout 1×10⁻⁷ to 5×10⁻⁴ g, or about 1×10⁻⁶ g to 5.0 g, or about 1×10⁻⁶ gto 2.5 g, or about 1×10⁻⁶ g to 1 g, or about 1×10⁻⁶ g to 0.5 g, or about1×10⁻⁶ g to 0.25 g, or about 1×10⁻⁶ g to 0.1 g, or about 1×10⁻⁶ g to5×10⁻² g, or about 1×10⁻⁶ to 5×10⁻² g, or about 1×10⁻⁶ g to 2.5×10⁻² g,or about 1×10⁻⁶ g to 1×10⁻² g, or about 1×10⁻⁶ g to 5×10⁻³ g, or about1×10⁻⁶ to 2.5×10⁻³ g, or about 1×10⁻⁶ g to 1×10⁻³ g, or about 1×10⁻⁶ gto 5×10⁻⁴ g, or about 1×10⁻⁵ g to 5 g, or about 1×10⁻⁵ g to 2.5 g, orabout 1×10⁻⁵ g to 1 g, or about 1×10⁻⁵ g to 0.5 g, or about 1×10⁻⁵ g to0.25 g, or about 1×10⁻⁵ g to 0.1 g, or about 1×10⁻⁵ g to 0.05 g, orabout 1×10⁻⁵ g to 2.5×10⁻² g, or about 1×10⁻⁵ g to 1×10⁻² g, or about1×10⁻⁵ g to 5×10⁻³ g, or about 1×10⁻⁵ g to 2.5×10⁻³ g, or about 1×10⁻⁵ gto 1×10⁻³ g, or about 1×10⁻⁵ g to 5×10⁴ g.

In some embodiments, the individual doses as described above, isrepeated 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times per day.

For other forms of administration, the daily dosages may range about therange described for systemic administration or may range about the rangedescribed for topical administration.

For slow or sustained release devices and formulations, in someembodiments, a typical dose range is about 0.1 mg to about 100 mg of theamorphous form or any of crystalline Forms A, B, C, D, or E, or acombination thereof, of the compound of Formula I, released over thedosing period. In other embodiments, about 1 mg to about 50 mg, about 1to about 25 mg, about 5 mg to about 100 mg, about 5 to about 50 mg,about 5 to about 25 mg, about 10 mg to about 100 mg, about 10 mg toabout 50 mg, about 10 mg to about 25 mg, or about 15 mg to about 50 mgis released over the dosing period. The dosing period for slow releasedevices and formulations, typically range from about 10 days to about 1year, about 30 days to about 1 year, about 60 days to about 1 year,about 3 months to about 1 year, about 4 months to about 1 year, about 5months to about 1 year, or about 6 months to about 1 year. In someembodiments, the slow release devices and formulations release theamorphous form or any of crystalline Forms A, B, C, D, or E, or acombination thereof, of the compound of Formula I, over the period ofabout 1 month to about 9 months, about 1 month to about 8 months, about1 month to about 7 months, about 1 month, to about 6 months, about 1month to about 5 months, about 1 month to about 4 months, or about 1month to about 3 months. In other embodiments the slow releaseformulations and devices release the amorphous form or any ofcrystalline Forms A, B, C, D, or E, or a combination thereof, of thecompound of Formula I, for up to 1 month, 2 months, 3 months, 4 months,5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 12 months,18 months, 2 years, 30 months, or 3 years. In some embodiments the slowor sustained release device is a periocular insert, intraocular insert,periocular implant, intraocular implant. In some embodiments, the slowor sustained release device is a pump. In some slow or sustained releaseformulations, it is a gel. In some slow or sustained releaseformulations, it is a biocompatible solid. In some slow or sustainedrelease formulations, it is a biodegradable solid.

In some embodiments of the invention, the sustained release formulationand/or implantations release sufficient therapeutic agent to sustain alocally effective level of therapeutic agent from the amorphous form orany of crystalline Forms A, B, C, D, or E, or a combination thereof, ofthe compound of Formula I, of at least about 10 nM, about 50 nM, about100 nM, about 150 nM, about 200 nM, about 250 nM, about 300 nM, about350 nM, about 500 nM, about 600 nM, about 700 nM, about 800 nM, about900 nM, about 1 μM, about 2 μM, about 3 μM, about 5 μM, about 6 μM,about 7 μM, about 8 μM, about 9 μM, about 10 μM, about 15 μM, about 20μM, about 25 μM, about 30 μM, about 35 μM, about 40 μM, about 45 μM,about 50 μM, about 55 μM, about 60 μM, about 65 μM, about 70 μM, about75 μM, about 80 μM, about 85 μM, about 90 μM, about 95 μM, or about 100μM across 1 year. In some embodiments of the invention, the sustainedrelease formulation and/or implantations release sufficient therapeuticagent intraocularly or periocularly to sustain a local level of theamorphous form or any of crystalline Forms A, B, C, D, or E, or acombination thereof, of the compound of Formula I, of at least about 10nM, about 50 nM, about 100 nM, about 150 nM, about 200 nM, about 250 nM,about 300 nM, about 350 nM, about 500 nM, about 600 nM, about 700 nM,about 800 nM, about 900 nM, about 1 μM, about 2 μM, about 3 μM, about 5μM, about 6 μM, about 7 μM, about 8 μM, about 9 μM, about 10 μM, about15 μM, about 20 μM, about 25 μM, about 30 μM, about 35 μM, about 40 μM,about 45 μM, about 50 μM, about 55 μM, about 60 μM, about 65 μM, about70 μM, about 75 μM, about 80 μM, about 85 μM, about 90 μM, about 95 μM,or about 100 μM across 6 months.

Formulations

The pharmaceutical compositions of the invention may be formulated as agel, cream, lotion, solution, suspension, emulsion, ointment, powder,crystalline forms, spray, aerosol, foam, salve, paste, plaster, paint,microparticle, nanoparticle, or bioadhesive, and may be prepared so asto contain liposomes, micelles and/or microspheres.

Formulations for topical use of the pharmaceutical compositions of thepresent invention can be provided as a topical composition wherein thepharmacologically active ingredients are mixed with excipients to form asemisolid consistency. Examples of such topical pharmaceuticalcompositions include, but are not limited to, a gel, cream, lotion,suspension, emulsion, ointment, foam, paste and the like. Alternatively,the topical pharmaceutical compositions of the present invention can beformulated in a semi-liquid formulation. Examples of such topicalpharmaceutical compositions include, but are not limited to, a topicalsolution, spray, mist, drops and the like. Alternatively, the topicalpharmaceutical compositions of the present invention can be formulatedin a dry powder form. The pharmaceutical compositions can also beadministered by a transdermal patch.

Ointments, as is well known in the art of pharmaceutical formulation,are semi-solid preparations that are typically based on petrolatum orother petroleum derivatives. As an ointment, the composition has aconsistency suitable for uniform dermal application. Additionally, theointment may be substantially viscous to remain in contact with the skinregardless of perspiration, excess moisture or environmental conditions.The specific ointment base to be used, as will be appreciated by thoseskilled in the art, is one that will provide for optimum drug delivery,and, preferably, will provide for other desired characteristics as well,e.g., emolliency or the like. As with other carriers or vehicles, anointment base should be inert, stable, nonirritating and nonsensitizing.As explained in Remington: The Science and Practice of Pharmacy, 19thEd. (Easton, Pa.: Mack Publishing Co., 1995), at pages 1399-1404,ointment bases may be grouped in four classes: oleaginous bases;emulsifiable-bases; emulsion bases; and water-soluble bases. Oleaginousointment bases include, for example, vegetable oils, fats obtained fromanimals, and semisolid hydrocarbons obtained from petroleum.Emulsifiable ointment bases, also known as absorbent ointment bases,contain little or no water and include, for example, hydroxystearinsulfate, anhydrous lanolin and hydrophilic petrolatum. Emulsion ointmentbases are either water-in-oil (W/O) emulsions or oil-in-water (O/W)emulsions, and include, for example, cetyl alcohol, glycerylmonostearate, lanolin, and stearic acid. Some water-soluble ointmentbases are prepared from polyethylene glycols of varying molecularweight; again, see Remington: The Science and Practice of Pharmacy forfurther information.

Creams, as also well known in the art, are viscous liquids or semi-solidemulsions, either oil-in-water or water-in-oil. Cream bases arewater-washable, and contain an oil phase, an emulsifier, and an aqueousphase. The oil phase, also called the “internal” phase, is generallycomprised of petrolatum and a fatty alcohol such as cetyl or stearylalcohol. The aqueous phase usually, although not necessarily, exceedsthe oil phase in volume, and generally contains a humectant. Theemulsifier in a cream formulation is generally a nonionic, anionic,cationic, or amphoteric surfactant.

Gels are semi-solid, suspension-type systems and are well known in theart. Gel forming agent for use herein can be any gelling agent typicallyused in the pharmaceutical art for topical semi solid dosage forms.Single-phase gels contain organic macromolecules distributedsubstantially uniformly throughout the carrier liquid, which istypically aqueous, but also can contain an alcohol and optionally anoil. In order to prepare a uniform gel, dispersing agents such asalcohol or glycerin can be added, or the gelling agent can be dispersedby tritration, mechanical mixing or stirring, or combinations thereof.The amount of gelling agents varies widely and will ordinarily rangefrom about 0.1% to about 2.0% by weight, based on the total weight ofthe composition. The gel forming agent also works by the principle ofcopolymerization. Under alkaline pH, carbomer in presence of waterundergoes cross linking and forms a gel like structure. The degree ofpolymerization is dependent upon the pH. At a threshold pH, theviscosities achieved by the polymer grade is the maximum.

Lotions, are preparations to be applied to the skin surface withoutfriction, and are typically semi-liquid preparations in which solidparticles, including the active agent, are present in a water or alcoholbase. Lotions are usually suspensions of solids, and comprise a liquidoily emulsion of the oil-in-water type. Lotions may be usefulformulations herein for treating large body areas, because of the easeof applying a more fluid composition. It desirable that the insolublematter in a lotion be finely divided. Lotions will typically containsuspending agents to produce better dispersions as well as compoundsuseful for localizing and holding the active agent in contact with theskin, e.g., methylcellulose, sodium carboxymethyl-cellulose, or thelike.

Pastes are semi-solid dosage forms in which the active agent issuspended in a suitable base. Depending on the nature of the base,pastes are divided between fatty pastes or those made from asingle-phase aqueous gels. The base in a fatty paste is generallypetrolatum or hydrophilic petrolatum or the like. The pastes made fromsingle-phase aqueous gels generally incorporate carboxymethylcelluloseor the like as a base.

Plasters are comprised of a pasty mixture that is spread on the body,either directly or after being saturated into a base material such ascloth. Medications, including the pharmacologically active bases of theinvention, may be dissolved or dispersed within the plaster to make amedicated plaster.

Bioadhesives are preparations that adhere to surfaces of body tissues.Polymeric bioadhesive formulations are well known in the art; see, forexample, Heller et al., “Biodegradable polymers as drug deliverysystems”, in Chasin, M. and Langer, R., eds.: Dekker, N.Y., pp. 121-161(1990); and U.S. Pat. No. 6,201,065. Suitable non-polymeric bioadhesivesare also known in the art, including certain fatty acid esters (U.S.Pat. No. 6,228,383).

The amorphous form or any of the crystalline forms of the compound ofFormula I or a combination thereof, may be formulated as a sterilesolution or suspension, in suitable vehicles, well known in the art.Suitable formulations and additional carriers and excipients aredescribed in Remington “The Science and Practice of Pharmacy” (20^(th)Ed., Lippincott Williams & Wilkins, Baltimore Md.), the teachings ofwhich are incorporated by reference in their entirety herein.

For injectable formulations, the vehicle may be chosen from those knownin art to be suitable, including aqueous solutions or oil suspensions,or emulsions, with sesame oil, corn oil, cottonseed oil, or peanut oil,as well as elixirs, mannitol, dextrose, or a sterile aqueous solution,and similar pharmaceutical vehicles. The formulation may also comprisepolymer compositions which are biocompatible, biodegradable, such aspoly(lactic-co-glycolic)acid. These materials may be made into micro ornanospheres, loaded with drug and further coated or derivatized toprovide superior sustained release performance. Vehicles suitable forperiocular or intraocular injection include, for example, suspensions oftherapeutic agent in injection grade water, liposomes and vehiclessuitable for lipophilic substances. Other vehicles for periocular orintraocular injection are well known in the art.

The concentration of drug may be adjusted, the pH of the solutionbuffered and the isotonicity adjusted to be compatible with intravenousinjection, as is well known in the art.

Oral formulations can be tablets, capsules, troches, pills, wafers,chewing gums, lozenges, aqueous solutions or suspensions, oilysuspensions, syrups, elixirs, or dispersible powders or granules, andthe like and may be made in any way known in the art. Oral formulationsmay also contain sweetening, flavoring, coloring and preservativeagents.

Intranasal administration may utilize an aerosol suspension ofrespirable particles comprised of the amorphous form or any of thecrystalline forms of the compound of Formula I, or a combinationthereof, along with an aerosol propellant which the individual inhales.The compound of the invention are absorbed into the lining of the lung.Alternatively, aerosol delivery to the eye may deliver the compound ofFormula I to the lacrimal tissues via nasolacrimal ducts, andsubsequently be delivered to the retinal tissues in a pharmaceuticallyeffective amount.

The pharmaceutical compositions may include one or more inertexcipients, which include water, buffered aqueous solutions,surfactants, volatile liquids, starches, polyols, granulating agents,microcrystalline cellulose, diluents, lubricants, acids, bases, salts,emulsions, such as oil/water emulsions, oils such as mineral oil andvegetable oil, wetting agents, chelating agents, antioxidants, sterilesolutions, complexing agents, disintegrating agents and the like. TheCTFA Cosmetic Ingredient Handbook, Seventh Edition, 1997 and the EighthEdition, 2000, which is incorporated by reference herein in itsentirety, describes a wide variety of cosmetic and pharmaceuticalingredients commonly used in skin care compositions, which are suitablefor use in the compositions of the present invention. Examples of thesefunctional classes disclosed in this reference include: absorbents,abrasives, anticaking agents, antifoaming agents, antimicrobial agents,antioxidants, binders, biological additives, buffering agents, bulkingagents, chelating agents, chemical additives, colorants, cosmeticastringents, cosmetic biocides, denaturants, drug astringents, externalanalgesics, film formers, fragrance components, humectants, opacifyingagents, pH adjusters, plasticizers, preservatives, reducing agents, skinbleaching agents, skin-conditioning agents (emollient, humectants,miscellaneous, and occlusive), skin protectants, solvents, foamboosters, hydrotropes, solubilizing agents, steroidal anti-inflammatoryagents, surfactants/emulsifying agents, suspending agents(nonsurfactant), sunscreen agents, topical analgesics, ultraviolet lightabsorbers, SPF boosters, thickening agents, waterproofing agents, andviscosity increasing agents (aqueous and nonaqueous).

Pharmaceutically acceptable excipients for tablet forms may comprisenontoxic ingredients such as inert diluents, such as calcium carbonate,sodium carbonate, lactose, calcium phosphate, or sodium phosphate, andthe like. In the case of tablets for oral use, carriers which arecommonly used include lactose and corn starch, and lubricating agentssuch as magnesium stearate are commonly added. For oral administrationin capsule form, useful carriers include lactose and corn starch.Further nonlimiting examples of carriers and excipients include milk,sugar, certain types of clay, gelatin, stearic acid or salts thereof,calcium stearate, talc, vegetable fats or oils, gums and glycols.

Surfactant which can be used to form pharmaceutical compositions anddosage forms of the invention include, but are not limited to,hydrophilic surfactants, lipophilic surfactants, and mixtures thereof.That is, a mixture of hydrophilic surfactants may be employed, a mixtureof lipophilic surfactants may be employed, or a mixture of at least onehydrophilic surfactant and at least one lipophilic surfactant may beemployed.

In some embodiments of the invention, the surfactant may be the sodiumsalt form of the compound, which may include the monosodium salt form.Suitable sodium salt surfactants may be selected based on desirableproperties, including high speed of polymerization, small resultantparticle sizes suitable for delivery, good polymerization yields,stability including freeze-thaw and shelf-life stability, improvedsurface tension properties, and lubrication properties.

The surfactant may be any suitable, non-toxic compound that isnon-reactive with the medicament and that substantially reduces thesurface tension between the medicament, the excipient and the site ofadministration. Some useful surfactants are: oleic acid available underthe tradenames Mednique 6322 and Emersol 6321 (from Cognis Corp.,Cincinnati, Ohio); cetylpyridinium chloride (from Arrow Chemical, Inc.Westwood, N.J.); soya lecithin available under the tradename Epikuron200 (from Lucas Meyer Decatur, Ill.); polyoxyethylene(20) sorbitanmonolaurate available under the tradename Tween 20 (from ICI SpecialtyChemicals, Wilmington, Del.); polyoxyethylene(20) sorbitan monostearateavailable under the tradename Tween 60 (from ICI); polyoxyethylene(20)sorbitan monooleate available under the tradename Tween 80 (from ICI);polyoxyethylene (10) stearyl ether available under the tradename Brij 76(from ICI); polyoxyethylene (2) oleyl ether available under thetradename Brij 92 (frown ICI);Polyoxyethylene-polyoxypropylene-ethylenediamine block copolymeravailable under the tradename Tetronic 150 R1 (from BASF);polyoxypropylene-polyoxyethylene block copolymers available under thetradenames Pluronic L-92, Pluronic L-121 end Pluronic F 68 (from BASF);castor oil ethoxylate available under the tradename Alkasurf CO-40 (fromRhone-Poulenc Mississauga Ontario, Canada); and mixtures thereof.

A suitable hydrophilic surfactant may generally have an HLB value of atleast 10, while suitable lipophilic surfactants may generally have anHLB value of or less than about 10. An empirical parameter used tocharacterize the relative hydrophilicity and hydrophobicity of non-ionicamphiphilic compounds is the hydrophilic-lipophilic balance (“HLB”value). Surfactants with lower HLB values are more lipophilic orhydrophobic, and have greater solubility in oils, while surfactants withhigher HLB values are more hydrophilic, and have greater solubility inaqueous solutions. Hydrophilic surfactants are generally considered tobe those compounds having an HLB value greater than about 10, as well asanionic, cationic, or zwitterionic compounds for which the HLB scale isnot generally applicable. Similarly, lipophilic (i.e., hydrophobic)surfactants are compounds having an HLB value equal to or less thanabout 10. However, HLB value of a surfactant is merely a rough guidegenerally used to enable formulation of industrial, pharmaceutical andcosmetic emulsions.

Hydrophilic surfactants may be either ionic or non-ionic. Suitable ionicsurfactants include, but are not limited to, alkylammonium salts;fusidic acid salts; fatty acid derivatives of amino acids,oligopeptides, and polypeptides; glyceride derivatives of amino acids,oligopeptides, and polypeptides; lecithins and hydrogenated lecithins;lysolecithins and hydrogenated lysolecithins; phospholipids andderivatives thereof; lysophospholipids and derivatives thereof;carnitine fatty acid ester salts; salts of alkylsulfates; fatty acidsalts; sodium docusate; acyl lactylates; mono- and di-acetylatedtartaric acid esters of mono- and di-glycerides; succinylated mono- anddi-glycerides; citric acid esters of mono- and di-glycerides; andmixtures thereof.

Within the aforementioned group, some ionic surfactants include, by wayof example: lecithins, lysolecithin, phospholipids, lysophospholipidsand derivatives thereof; carnitine fatty acid ester salts; salts ofalkylsulfates; fatty acid salts; sodium docusate; acyl lactylates; mono-and di-acetylated tartaric acid esters of mono- and di-glycerides;succinylated mono- and di-glycerides; citric acid esters of mono- anddi-glycerides; and mixtures thereof.

Ionic surfactants may be the ionized forms of lecithin, lysolecithin,phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol,phosphatidic acid, phosphatidylserine, lysophosphatidylcholine,lysophosphatidylethanolamine, lysophosphatidyiglycerol, lysophosphatidicacid, lysophosphatidylserine, PEG-phosphatidylethanolamine,PVP-phosphatidylethanolamine, lactylic esters of fatty acids,stearoyl-2-lactylate, stearoyl lactylate, succinylated monoglycerides,mono/diacetylated tartaric acid esters of mono/diglycerides, citric acidesters of mono/diglycerides, cholylsarcosine, caproate, caprylate,caprate, laurate, myristate, palmitate, oleate, ricinoleate, linoleate,linolenate, stearate, lauryl sulfate, teracecyl sulfate, docusate,lauroyl carnitines, palmitoyl carnitines, myristoyl camitines, and saltsand mixtures thereof.

Hydrophilic non-ionic surfactants may include, but not limited to,alkylglucosides; alkylmaltosides; alkylthioglucosides; laurylmacrogolglycerides; polyoxyalkylene alkyl ethers such as polyethyleneglycol alkyl ethers; polyoxyalkylene alkylphenols such as polyethyleneglycol alkyl phenols; polyoxyalkylene alkyl phenol fatty acid esterssuch as polyethylene glycol fatty acids monoesters and polyethyleneglycol fatty acids diesters; polyethylene glycol glycerol fatty acidesters; polyglycerol fatty acid esters; polyoxyalkylene sorbitan fattyacid esters such as polyethylene glycol sorbitan fatty acid esters;hydrophilic transesterification products of a polyol with at least onemember of the group consisting of glycerides, vegetable oils,hydrogenated vegetable oils, fatty acids, and sterols; polyoxyethylenesterols, derivatives, and analogues thereof; polyoxyethylated vitaminsand derivatives thereof; polyoxyethylene-polyoxypropylene blockcopolymers; and mixtures thereof; polyethylene glycol sorbitan fattyacid esters and hydrophilic transesterification products of a polyolwith at least one member of the group consisting of triglycerides,vegetable oils, and hydrogenated vegetable oils. The polyol may beglycerol, ethylene glycol, polyethylene glycol, sorbitol, propyleneglycol, pentaerythritol, or a saccharide.

Other hydrophilic-non-ionic surfactants include, without limitation,PEG-10 laurate, PEG-12 laurate, PEG-20 laurate, PEG-32 laurate, PEG-32dilaurate, PEG-12 oleate, PEG-15 oleate, PEG-20 oleate, PEG-20 dioleate,PEG-32 oleate, PEG-200 oleate, PEG-400 oleate, PEG-15 stearate, PEG-32distearate, PEG-40 stearate, PEG-100 stearate, PEG-20 dilaurate, PEG-25glyceryl triolcate, PEG-32 dioleate, PEG-20 glyceryl laurate, PEG-30glyceryl laurate, PEG-20 glyceryl stearate, PEG-20 glyceryl oleate,PEG-30 glyceryl oleate, PEG-30 glyceryl laurate, PEG-40 glyceryllaurate, PEG-40 palm kernel oil, PEG-50 hydrogenated castor oil, PEG-40castor oil, PEG-35 castor oil, PEG-60 castor oil, PEG-40 hydrogenatedcastor oil, PEG-60 hydrogenated castor oil, PEG-60 corn oil, PEG-6caprate/caprylate glycerides, PEG-8 caprate/caprylate glycerides,polyglyceryl-10 laurate, PEG-30 cholesterol, PEG-25 phyto sterol, PEG-30soya sterol, PEG-20 trioleate, PEG-40 sorbitan oleate, PEG-80 sorbitanlaurate, polysorbate 20, polysorbate 80, POE-9 lauryl ether, POE-23lauryl ether, POE-10 oleyl ether, POE-20 oleyl ether, POE-20 stearylether, tocopheryl PEG-100 succinate, PEG-24 cholesterol,polyglyceryl-10oleate, Tween 40, Tween 60, sucrose monostearate, sucrosemonolaurate, sucrose monopalmitate, PEG 10-100 nonyl phenol series, PEG15-100 octyl phenol series, and poloxamers.

Suitable lipophilic surfactants include, by way of example only: fattyalcohols; glycerol fatty acid esters; acetylated glycerol fatty acidesters; lower alcohol fatty acids esters; propylene glycol fatty acidesters; sorbitan fatty acid esters; polyethylene glycol sorbitan fattyacid esters; sterols and sterol derivatives; polyoxyethylated sterolsand sterol derivatives; polyethylene glycol alkyl ethers; sugar esters;sugar ethers; lactic acid derivatives of mono- and di-glycerides;hydrophobic transesterification products of a polyol with at least onemember of the group consisting of glycerides, vegetable oils,hydrogenated vegetable oils, fatty acids and sterols; oil-solublevitamins/vitamin derivatives; and mixtures thereof. Within this group,some lipophilic surfactants include glycerol fatty acid esters,propylene glycol fatty acid esters, and mixtures thereof, or arehydrophobic transesterification products of a polyol with at least onemember of the group consisting of vegetable oils, hydrogenated vegetableoils, and triglycerides.

Surfactants may be used in any formulation of the invention where itsuse is not otherwise contradicted. In some embodiments of the invention,the use of no surfactants or limited classes of surfactants may bepreferred. The topical formulations according to the invention maycontain no, or substantially no surfactant, i.e. contain less thanapproximately 0.0001% by weight of surface-active agents. If desired,however, the formulations can contain surface-active agentsconventionally employed in topical formulations, such as oleic acid,lecithin, sorbitan trioleate, cetylpyridinium chloride, benzalkoniumchloride, polyoxyethylene (20) sorbitan monolaurate, polyoxyethylene(20) sorbitan monostearate, polyoxyethylene (20) sorbitan mono-oleate,polyoxypropylene/polyoxyethylene block copolymers,polyoxypropylene/polyoxyethylene/ethylenediamine block copolymers,ethoxylated castor oil and the like, where the proportion ofsurface-active agents, if present, can be about 0.0001 to 1% by weight,or about 0.001 to 0.1% by weight, based on the total formulation. Othersuitable surfactant/emulsifying agents would be known to one of skill inthe art and are listed in the CTFA International Cosmetic IngredientDictionary and Handbook, Vol. 2, 7th Edition (1997).

Other suitable aqueous vehicles include, but are not limited to,Ringer's solution and isotonic sodium chloride. Aqueous suspensions mayinclude suspending agents such as cellulose derivatives, sodiumalginate, polyvinyl-pyrrolidone and gum tragacanth, and a wetting agentsuch as lecithin. Suitable preservatives for aqueous suspensions includeethyl and n-propyl p-hydroxybenzoate.

Chelating agents which can be used to form pharmaceutical compositionsand dosage forms of the invention include, but are not limited to,ethylene diaminetetraacetic acid (EDTA), EDTA disodium, calcium disodiumedetate, EDTA trisodium, albumin, transferrin, desferoxamine, desferal,desferoxamine mesylate, EDTA tetrasodium and EDTA dipotassium, sodiummetasilicate or combinations of any of these.

Preservatives which can be used to form pharmaceutical compositions anddosage forms of the invention include, but are not limited to, purite,peroxides, perborates, imidazolidinyl urea, diazolidinyl urea,phenoxyethanol, alkonium chlorides including benzalkonium chlorides,methylparaben, ethylparaben and propylparaben. In other embodiments,suitable preservatives for the compositions of the invention include:benzalkonium chloride, purite, peroxides, perborates, thimerosal,chlorobutanol, methyl paraben, propyl paraben, phenylethyl alcohol,edetate disodium, sorbic acid, Onamer M, or other agents known to thoseskilled in the art. In some embodiments of the invention, suchpreservatives may be employed at a level of from 0.004% to 0.02% W/V. Insome compositions of the present application the preservative, forexample, benzalkonium chloride, methyl paraben, and/or propyl paraben,may be employed at a level of from about 0.001% to less than about0.01%, e.g. from about 0.001% to about 0.008%, or about 0.005% W/V. Ithas been found that a concentration of benzalkonium chloride of about0.005% may be sufficient to preserve the compositions of the presentinvention from microbial attack. One of skill in the art could determinethe proper concentration of ingredients as well as combinations ofvarious ingredients for generating a suitable topical formulation. Forexample, ophthalmic drops or formulations for application to skin mayuse a mixture of methyl and propyl parabens at about 0.02% W/V and about0.04% W/V respectively. In some embodiments, these formulations usemethyl paraben and/or propyl paraben in amounts up to about 0.02% W/Vand up to about 0.04% W/V respectively, which encompasses theembodiments where no methyl paraben or no propyl paraben is used.

Lubricants which can be used to form pharmaceutical compositions anddosage forms of the invention include, but are not limited to, calciumstearate, magnesium stearate, mineral oil, light mineral oil, glycerin,sorbitol, mannitol, polyethylene glycol, other glycols, stearic acid,sodium lauryl sulfate, talc, hydrogenated vegetable oil (e.g., peanutoil, cottonseed oil, sunflower oil, sesame oil, olive oil, corn oil, andsoybean oil), zinc stearate, ethyl oleate, ethyl laureate, agar, ormixtures thereof. Additional lubricants include, for example, a syloidsilica gel, a coagulated aerosol of synthetic silica, or mixturesthereof. A lubricant can optionally be added, in an amount of less thanabout 1 weight percent of the pharmaceutical composition.

Thickening agents which can be used to form pharmaceutical compositionsand dosage forms of the invention include, but are not limited to,isopropyl myristate, isopropyl palmitate, isodecyl neopentanoate,squalene, mineral oil, C₁₂-C₁₅ benzoate and hydrogenated polyisobutene.Agents which would not disrupt other compounds of the final product maybe desirable, such as non-ionic thickening agents. The selection ofadditional thickening agents is well within the skill of one in the art.

Skin conditioning agents can be emollients, humectants and moisturizers.A humectant is a moistening agent that promotes retention of water dueto its hygroscopic properties. Suitable skin conditioning agents includeurea; guanidine; aloe vera; glycolic acid and glycolate salts such asammonium and quaternary alkyl ammonium; lactic acid and lactate saltssuch as sodium lactate, ammonium lactate and quaternary alkyl ammoniumlactate; polyhydroxy alcohols such as sorbitol, glycerol, mannitol,xylitol, hexanetriol, propylene glycol, butylene glycol, hexyleneglycol, polymeric glycols such as polyethylene glycol and polypropyleneglycol; carbohydrates such as alkoxylated glucose; starches; starchderivatives; glycerin; pyrrolidone carboxylic acid (PCA); lactamidemonoethanolamine; acetamide monoethanolamine; volatile silicone oils;nonvolatile silicone oils; and mixtures thereof. Suitable silicone oilscan be polydialkylsiloxanes, polydiarylsiloxanes, polyalkarylsiloxanesand cyclomethicones having 3 to 9 silicon atoms.

An emollient is an oleaginous or oily substance which helps to smoothand soften the skin, and may also reduce its roughness, cracking orirritation. Typical suitable emollients include mineral oil having aviscosity in the range of 50 to 500 centipoise (cps), lanolin oil,coconut oil, cocoa butter, olive oil, almond oil, macadamia nut oil,aloe extracts such as aloe vera lipoquinone, synthetic jojoba oils,natural sonora jojoba oils, safflower oil, corn oil, liquid lanolin,cottonseed oil and peanut oil. In some embodiments, the emollient is acocoglyceride, which is a mixture of mono, di and triglycerides of cocoaoil, sold under the trade name of Myritol 331 from Henkel KGaA, orDicaprylyl Ether available under the trade name Cetiol OE from HenkelKGaA or a C.sub.12-C.sub.15 Alkyl Benzoate sold under the trade nameFinsolv TN from Finetex. Another suitable emollient is DC 200 Fluid 350,a silicone fluid, available Dow Corning Corp.

Other suitable emollients include squalane, castor oil, polybutene,sweet almond oil, avocado oil, calophyllum oil, ricin oil, vitamin Eacetate, olive oil, silicone oils such as dimethylopolysiloxane andcyclomethicone, linolenic alcohol, oleyl alcohol, the oil of cerealgerms such as the oil of wheat germ, isopropyl palmitate, octylpalmitate, isopropyl myristate, hexadecyl stearate, butyl stearate,decyl oleate, acetyl glycerides, the octanoates and benzoates of(C.sub.12-C.sub.15) alcohols, the octanoates and decanoates of alcoholsand polyalcohols such as those of glycol and glyceryl, ricinoleatesesters such as isopropyl adipate, hexyl laurate and octyl dodecanoate,dicaprylyl maleate, hydrogenated vegetable oil, phenyltrimethicone,jojoba oil and aloe vera extract.

Other suitable emollients which are solids or semi-solids at ambienttemperatures may be used. Such solid or semi-solid cosmetic emollientsinclude glyceryl dilaurate, hydrogenated lanolin, hydroxylated lanolin,acetylated lanolin, petrolatum, isopropyl lanolate, butyl myristate,cetyl myristate, myristyl myristate, myristyl lactate, cetyl alcohol,isostearyl alcohol and isocetyl lanolate. One or more emollients canoptionally be included in the formulation.

Anti-oxidants which can be used to form pharmaceutical compositions anddosage forms of the invention include, but are not limited to, propyl,octyl and dodecyl esters of gallic acid, butylated hydroxyanisole (BHA,usually purchased as a mixture of ortho and meta isomers), green teaextract, uric acid, cysteine, pyruvate, nordihydroguaiaretic acid,ascorbic acid, salts of ascorbic acid such as ascorbyl palmitate andsodium ascorbate, ascorbyl glucosamine, vitamin E (i.e., tocopherolssuch as a-tocopherol), derivatives of vitamin E (e.g., tocopherylacetate), retinoids such as retinoic acid, retinol, trans-retinol,cis-retinol, mixtures of trans-retinol and cis-retinol, 3-dehydroretinoland derivatives of vitamin A (e.g., retinyl acetate, retinal and retinylpalmitate, also known as tetinyl palmitate), sodium citrate, sodiumsulfite, lycopene, anthocyanids, bioflavinoids (e.g., hesperitin,naringen, rutin and quercetin), superoxide dismutase, glutathioneperoxidase, butylated hydroxytoluene (BHT), indole-3-carbinol,pycnogenol, melatonin, sulforaphane, pregnenolone, lipoic acid and4-hydroxy-5-methyl-3[2H]-furanone.

Skin protecting agents are agents that protect the skin against chemicalirritants and/or physical irritants, e.g., UV light, includingsunscreens, anti-acne additives, anti-wrinkle and anti-skin atrophyagents. Suitable sunscreens as skin protecting agents include2-ethylhexyl p-methoxycinnamate, 2-ethylhexylN,N-dimethyl-p-aminobenzoate, p-aminobenzoic acid,2-phenylbenzimidazole-5-sulfonic acid, octocrylene, oxybenzone,homomenthyl salicylate, octyl salicylate,4,4′-methoxy-t-butyldibenzoylmethane, 4-isopropy dibenzoylmethane,3-benzylidene camphor, 3-(4-methylbenzylidene) camphor, anthanilates,ultrafine titanium dioxide, zinc oxide, iron oxide, silica,4-N,N-(2-ethylhexyl)methylaminobenzoic acid ester of2,4-dihydroxybenzophenone, 4-N,N-(2-ethylhexyl)-methylaminobenzoic acidester with 4-hydroxydibenzoylmethane,4-N,N-(2-ethylhexyl)-methylaminobenzoic acid ester of2-hydroxy-4-(2-hydroxyethoxy)benzophenone and4-N,N(2-ethylhexyl)-methylaminobenzoic acid ester of4-(2-hydroxyethoxy)dibenzoylmethane. Suitable anti-acne agents includesalicylic acid; 5-octanoyl salicylic acid; resorcinol; retinoids such asretinoic acid and its derivatives; sulfur-containing D and L amino acidsother than cysteine; lipoic acid; antibiotics and antimicrobials such asbenzoyl peroxide, octopirox, tetracycline,2,4,4′-trichloro-2′-hydroxydiphenyl ether, 3,4,4′-trichlorobanilide,azelaic acid, phenoxyethanol, phenoxypropenol, phenoxisopropanol, ethylacetate, clindamycin and melclocycline; flavonoids; and bile salts suchas scymnol sulfate, deoxycholate and cholate. Examples of anti-wrinkleand anti-skin atrophy agents are retinoic acid and its derivatives,retinol, retinyl esters, salicylic acid and its derivatives,sulfur-containing D and L amino acids except cysteine, alpha-hydroxyacids (e.g., glycolic acid and lactic acid), phytic acid, lipoic acidand lysophosphatidic acid.

The formulations may also contain irritation-mitigating additives tominimize or eliminate the possibility of skin irritation or skin damageresulting from the permeation-enhancing base or other components of thecomposition. Suitable irritation-mitigating additives include, forexample: -tocopherol; monoamine oxidase inhibitors, particularly phenylalcohols such as 2-phenyl-1-ethanol; glycerin; salicylic acids andsalicylates; ascorbic acids and ascorbates; ionophores such as monensin;amphiphilic amines; ammonium chloride; N-acetylcysteine; cis-urocanicacid; capsaicin; and chloroquine. The irritant-mitigating additive, ifpresent, may be incorporated into the present formulations at aconcentration effective to mitigate irritation or skin damage, typicallyrepresenting not more than about 20 wt. %, more typically not more thanabout 5 wt. %, of the composition.

A dry-feel modifier is an agent which when added to an emulsion, impartsa “dry feel” to the skin when the emulsion dries. Dry feel modifiers caninclude talc, kaolin, chalk, zinc oxide, silicone fluids, inorganicsalts such as barium sulfate, surface treated silica, precipitatedsilica, fumed silica such as an Aerosil available from Degussa Inc. ofNew York, N.Y. U.S.A. Another dry feel modifier is an epichlorohydrincross-linked glyceryl starch of the type that is disclosed in U.S. Pat.No. 6,488,916.

Other agents may also be added, such as antimicrobial agents, to preventspoilage upon storage, i.e., to inhibit growth of microbes such asyeasts and molds. Suitable antimicrobial agents are typically selectedfrom the group consisting of the methyl and propyl esters ofp-hydroxybenzoic acid (i.e., methyl and propyl paraben), sodiumbenzoate, sorbic acid, imidurea, purite, peroxides, perborates andcombinations thereof.

The formulation may also contain an aesthetic agent. Examples ofaesthetic agents include fragrances, pigments, colorants, essentialoils, skin sensates and astringents. Suitable aesthetic agents includeclove oil, menthol, camphor, eucalyptus oil, eugenol, methyl lactate,bisabolol, witch hazel distillate and green tea extract.

Fragrances are aromatic substances which can impart an aestheticallypleasing aroma to the composition. Typical fragrances include aromaticmaterials extracted from botanical sources (i.e., rose petals, gardeniablossoms, jasmine flowers, etc.) which can be used alone or in anycombination to create essential oils. Alternatively, alcoholic extractsmay be prepared for compounding fragrances. However, due to therelatively high costs of obtaining fragrances from natural substances,the modern trend is to use synthetically prepared fragrances,particularly in high-volume products. One or more fragrances canoptionally be included in the composition in an amount ranging fromabout 0.001 to about 5 weight percent, or about 0.01 to about 0.5percent by weight. Additional preservatives may also be used if desiredand include well known preservative compositions such as benzyl alcohol,phenyl ethyl alcohol and benzoic acid, diazolydinyl, urea,chlorphenesin, iodopropynyl and butyl carbamate, among others.

In order to increase the degree and rate at which a drug penetrates theskin when delivering the compound of Formula I via topicaladministration, various approaches have been followed, each of whichinvolves the use of either a chemical penetration enhancer or a physicalpenetration enhancer. Physical enhancements of skin permeation include,for example, electrophoretic techniques such as iontophoresis. The useof ultrasound (or “phonophoresis”) as a physical penetration enhancerhas also been researched. Chemical penetration enhancers are morecommonly used. These are compounds that are topically administered alongwith a drug (or, in some cases, prior to drug administration) in orderto increase the permeability of the stratum corneum, and thereby providefor enhanced penetration of the drug through the skin. Ideally, suchchemical penetration enhancers (or “permeation enhancers,” as thecompounds are referred to herein) are compounds that are innocuous andserve merely to facilitate diffusion of the drug through the stratumcorneum.

Various compounds for enhancing the permeability of skin are known inthe art. Compounds that have been used to enhance skin permeabilityinclude: sulfoxides such as dimethylsulfoxide (DMSO) anddecylmethylsulfoxide (C₁₀MSO); ethers such as diethylene glycolmonoethyl ether (available commercially as Transcutol®) and diethyleneglycol monomethyl ether, surfactants such as sodium laurate, sodiumlauryl sulfate, ectyltrimethylammonium bromide, benzalkonium chloride,Poloxamer (231, 182, 184), Tween (20, 40, 60, 80), and lecithin (U.S.Pat. No. 4,783,450); the 1-substituted azacycloheptan-2-ones,particularly 1-n-dodecylcyclazacycloheptan-2-one (available under thetrademark Azone® from Nelson Research & Development Co., Irvine, Calif.;see U.S. Pat. Nos. 3,989,816, 4,316,893, 4,405,616, and 4,557,934);alcohols such as ethanol, propanol, octanol, benzyl alcohol, and thelike; fatty acids such as lauric acid, oleic acid and valeric acid;fatty acid esters such as isopropyl myristate, isopropyl palmitate,methylpropionate, and ethyl oleate; polyols and esters thereof such aspropylene glycol, ethylene glycol, glycerol, butanediol, polyethyleneglycol, and polyethylene glycol monolaurate (PEGML; see, e.g., U.S. Pat.No. 4,568,343); amides and other nitrogenous compounds such as urea,dimethylacetamide (DMA), dimethylformamide (DMF), 2-pyrrolidone,1-methyl-2-pyrrolidone, ethanolamine, diethanolamine andtriethanolamine; terpenes; alkanones; and organic acids, particularlysalicylic acid and salicylates, citric acid, and succinic acid. (SeePercutaneous Penetration Enhancers, Smith et al., editors, CRC Press,1995, for a number of chemical and physical enhancers.)

It has long been thought that strong bases, such as NaOH, were notsuitable as permeation enhancers because they would damage skin. It hasbeen now been discovered that the skin permeability of various drugscould be enhanced without skin damage by exposing the skin to a base orbasic solution, in a skin contacting formulation or patch. The desiredpH of the solution on the skin can be obtained using a variety of basesor base concentrations. Accordingly, the pH is selected so as to be lowenough so as to not cause skin damage, but high enough to enhance skinpermeation to various active agents. As such, it is important that theamount of base in any patch or formulation is optimized so as toincrease the flux of the drug through the body surface while minimizingany possibility of skin damage. In general, this means that the pH atthe body surface in contact with a formulation or drug delivery systemof the invention may be in the range of approximately 8.0-13.0, about8.0-11.5, about 8.5 to 11.5, or about 8.5-10.5. In some embodiments, thepH will be in the range of about 9.5 to 11.5, or about 10.0 to 11.5.

In one embodiment, the pH at the skin surface is the primary designconsideration, i.e., the composition or system is designed so as toprovide the desired pH at the skin surface. Anhydrous formulations andtransdermal systems may not have a measurable pH, and the formulation orsystem can be designed so as to provide a target pH at the skin surface.Moisture from the body surface can migrate into the formulation orsystem, dissolve the base and thus release the base into solution, whichwill then provide the desired target pH at body surface. In thoseinstances, a hydrophilic composition may be preferred. In addition, whenusing aqueous formulations, the pH of the formulation may change overtime after it is applied on the skin. For example, gels, solutions,ointments, etc., may experience a net loss of moisture after beingapplied to the body surface, i.e., the amount of water lost is greaterthan the amount of water received from the body surface. In that case,the pH of the formulation may be different than its pH whenmanufactured. This problem can be easily remedied by designing theaqueous formulations to provide a target pH at the body surface.

In other embodiments of the invention, the pH of the formulation or thedrug composition contained within a delivery system will be in the rangeof approximately pH 8.0 to about pH 13.0, about pH 8.0 to about pH 11.5,about pH 8.5 to about pH 11.5, or about pH 8.5 to about pH 10.5. In someembodiments, the pH will be in the range of about pH 9.5 to about pH11.5, or about pH 10.0 to about pH 11.5. In one embodiment of theinvention the pH of the formulation is higher than the pH at the bodysurface. For example, if an aqueous formulation is used, moisture fromthe body surface can dilute the formulation, and thus provide for adifferent pH at the body surface, which will typically be lower thanthat of the formulation itself.

In one embodiment, the body surface is exposed to a base or basicsolution for a sufficient period of time so as to provide a high pH atthe skin surface, thus creating channels in the skin or mucosa for thedrug to go through. It is expected that drug flux is proportional to thestrength of the solution and the duration of exposure. However, it isdesirable to balance the maximization of drug flux with the minimizationof skin damage. This can be done in numerous ways. For example, the skindamage may be minimized by selecting a lower pH within the 8.0 to 13.0range, by exposing the skin to the formulation or system for a shorterperiod of time, or by including at least one irritation-mitigatingadditive. Alternatively, the patient can be advised to change thelocation of application with each subsequent administration.

While certain amounts are set forth below, it is understood that, forall of the inorganic and organic bases described herein, the optimumamount of any such base will depend on the strength or weakness of thebase and its molecular weight, and other factors such as the number ofionizable sites in the active agent being administered and whether thereare any acidic species present in the formulation or patch. One skilledin the art may readily determine the optimum amount for any particularbase such that the degree of enhancement is optimized while thepossibility of damage to the body surface is eliminated or at leastsubstantially minimized.

Exemplary inorganic bases are inorganic hydroxides, inorganic oxides,inorganic salts of weak acids, and combinations thereof. Some usefulinorganic bases include those whose aqueous solutions have a high pH,and are acceptable as food or pharmaceutical additives. Examples of suchinorganic bases include ammonium hydroxide, sodium hydroxide, potassiumhydroxide, calcium hydroxide, magnesium hydroxide, magnesium oxide,calcium oxide, Ca(OH)₂, sodium acetate, sodium borate, sodiummetaborate, sodium carbonate, sodium bicarbonate, sodium phosphate,potassium carbonate, potassium bicarbonate, potassium citrate, potassiumacetate, potassium phosphate and ammonium phosphate and combinationsthereof.

Inorganic hydroxides include, for example, ammonium hydroxide, alkalimetal hydroxide and alkaline earth metal hydroxides, and mixturesthereof. Some useful inorganic hydroxides include ammonium hydroxide;monovalent alkali metal hydroxides such as sodium hydroxide andpotassium hydroxide; divalent alkali earth metal hydroxides such ascalcium hydroxide and magnesium hydroxide; and combinations thereof.

The amount of inorganic hydroxide included in the compositions andsystems of the invention, will typically represent about 0.3-7.0 W/V %,about 0.5-4.0 W/V %, about 0.5-3.0 W/V %, or about 0.75-2.0 W/V %, of atopically applied formulation or of a drug reservoir of a drug deliverysystem, or patch.

Inorganic oxides include, for example, magnesium oxide, calcium oxide,and the like.

The amount of inorganic oxide included in the compositions and systemsof the invention may be substantially higher than the numbers set forthabove for the inorganic hydroxide, and may be as high as 20 wt %, insome cases as high as 25 wt % or higher, but will generally be in therange of about 2-20 wt %. These amounts may be adjusted to take intoconsideration the presence of any base-neutralizable species.

Inorganic salts of weak acids include, ammonium phosphate (dibasic);alkali metal salts of weak acids such as sodium acetate, sodium borate,sodium metaborate, sodium carbonate, sodium bicarbonate, sodiumphosphate (tribasic), sodium phosphate (dibasic), potassium carbonate,potassium bicarbonate, potassium citrate, potassium acetate, potassiumphosphate (dibasic), potassium phosphate (tribasic); alkaline earthmetal salts of weak acids such as magnesium phosphate and calciumphosphate; and the like, and combinations thereof.

Organic bases suitable for use in the invention are compounds having anamino group, amido group, an oxime, a cyano group, an aromatic ornon-aromatic nitrogen-containing heterocycle, a urea group, andcombinations thereof. More specifically, examples of suitable organicbases are nitrogenous bases, which include, but are not limited to,primary amines, secondary amines, tertiary amines, amidines, guanidines,hydroxylamines, cyano guanidines, cyanoamidines, oximes, cyano (—CN)containing groups, aromatic and non-aromatic nitrogen-containingheterocycles, urea, and mixtures thereof. In some embodiments, theorganic bases are primary amines, secondary amines, tertiary amines,aromatic and non-aromatic nitrogen-containing heterocycles, and mixturesthereof.

For all permeation-enhancing bases herein, the optimum amount of anyparticular agent will depend on the strength or weakness of the base,the molecular weight of the base, and other factors such as the numberof ionizable sites in the drug administered and any other acidic speciesin the formulation or patch. One skilled in the art may readilydetermine the optimum amount for any particular agent by ensuring that aformulation is effective to provide a pH at the skin surface, uponapplication of the formulation, in the range of about pH 7.5 to about pH13.0, about pH 8.0 to about pH 11.5, or about pH 8.5 to about pH 10.5.In some embodiments, the pH will be in the range of about pH 9.5 toabout pH 11.5, or about pH 10.0 to about pH 11.5. This in turn ensuresthat the degree of treatment is maximized while the possibility ofdamage to the body surface is eliminated or at least substantiallyminimized.

In the case of intranasal administration, such solutions or suspensionsmay be isotonic relative to nasal secretions and of about the same pH,ranging e.g., from about pH 4.0 to about pH 7.4 or, from about pH 6.0 toabout pH 7.0. Buffers should be physiologically compatible and include,simply by way of example, phosphate buffers. For example, arepresentative nasal decongestant is described as being buffered to a pHof about 6.2 (Remington's Pharmaceutical Sciences 16th edition, Ed.Arthur Osol, page 1445 (1980)). One skilled in the art can readilydetermine a suitable saline content and pH for an innocuous aqueoussolution for nasal and/or upper respiratory administration. An exampleof a suitable formulation for intranasal administration, is an aqueoussolution buffered to a pH of about 6.0 to about 8.0 with SodiumPhosphate, Monobasic, comprising about 1% W/V of the LFA-1 antagonist,up to about 0.1% W/V EDTA, and, optionally, up to about 0.4% w/wMethylparaben and up to about 0.02% w/w Propylparaben.

Additional permeation enhancers will be known to those of ordinary skillin the art of topical drug delivery, and/or are described in thepertinent texts and literature. See, e.g., Percutaneous PenetrationEnhancers, Smith et al., eds. (CRC Press, 1995).

It is envisioned additionally, that the amorphous form or any of thecrystalline forms of the compound of Formula I, or a combinationthereof, may be attached releasably to biocompatible polymers for use insustained release formulations on, in or attached to inserts fortopical, intraocular, periocular, or systemic administration. Thecontrolled release from a biocompatible polymer may be utilized with awater soluble polymer to form an instillable formulation, as well. Thecontrolled release from a biocompatible polymer, such as for example,PLGA microspheres or nanospheres, may be utilized in a formulationsuitable for intra ocular implantation or injection for sustainedrelease administration, as well. Any suitable biodegradable andbiocompatible polymer may be used.

Additionally, the amorphous form or any of the crystalline Forms A, B,C, D, and E, or combinations thereof, of the compound of Formula I maybe suitable for use in sustained release formulations where the drugentity may remain as a solid. Further, the calcium salt of the free acidof any of these forms is envisioned to be useful in slow releaseformulations, as a solid formulation, gel formulation or liquidformulation.

The amorphous form or any of the crystalline Forms A, B, C, D, and E, orcombinations thereof, of the compound of Formula I may be milled toprovide more suitable properties for formulation. Milling may providesmaller particle size with greater surface area exposure, which canprovide faster solubilization in-vivo or during formulation.Alternatively, milling to a smaller particle size may provide theamorphous form or any of the crystalline Forms A, B, C, D, or E, or acombination thereof, with the capacity to pass through biologicalbarriers, such as the skin or gut wall, directly, without initialsolubilization, permitting the use of the amorphous form or any of thecrystalline Forms A, B, C, D, and E, or combinations thereof, as a solidin the formulation, which may provide additional benefits of temperaturestability, shelf life, ease of transport, and ease of use by thesubject. Milled solid particles of the amorphous form or any of thecrystalline Forms A, B, C, D, and E, or combinations thereof, may alsoprovide greater bioavailability, and more desirable or controllablepharmacokinetics in the formulations. The size of the milled particlecan affect the rate of distribution of the compound of Formula I uponadministration or rate of release of the compound of Formula I from asustained or slow release formulation. Further, milling of the particlesof the amorphous form or any of the crystalline Forms A, B, C, D, and E,or combinations thereof, may be performed to create either a narrower ormore symmetrical particle size distribution within a particularformulation or lot of material which may be subjected to formulation.The size of the particles of the amorphous form or any of thecrystalline Forms A, B, C, D, and E, or combinations thereof, may beselected as is well known in the art, to obtain the desired physicalcharacteristics for ease of formulation or the ability to be distributedfrom the formulation in a controlled fashion over a preselected periodunder conditions of use. The size of the particles can be represented asthe D50, which represents the median or 50^(th) percentile of thediameter of a particle within the lot of material under discussion.Another measure of the size of the particles in a lot of material is theD90, which is the 90^(th) percentile of the particle size diameter inthe particle size distribution.

For transdermal administration, any suitable formulation known in theart may be utilized, either as a solution, suspension, gel, powder,cream, oil, solids, dimethylsulfoxide (DMSO)-based solutions orliposomal formulation for use in a patch or other delivery system knownin the art. The pharmaceutical compositions also may comprise suitablesolid or gel phase carriers or excipients, which are compounds thatallow increased penetration of, or assist in the delivery of,therapeutic molecules across the stratum corneum permeability barrier ofthe skin. There are many of these penetration-enhancing molecules knownto those trained in the art of topical formulation. Examples of suchcarriers and excipients include, but are not limited to, humectants(e.g., urea), glycols (e.g., propylene glycol), alcohols (e.g.,ethanol), fatty acids (e.g., oleic acid), surfactants (e.g., isopropylmyristate and sodium lauryl sulfate), pyrrolidones, glycerolmonolaurate, sulfoxides, terpenes (e.g., menthol), amines, amides,alkanes, alkanols, water, calcium carbonate, calcium phosphate, varioussugars, starches, cellulose derivatives, gelatin, and polymers such aspolyethylene glycols. The construction and use of transdermal patchesfor the delivery of pharmaceutical agents is well known in the art. See,e.g., U.S. Pat. Nos. 5,023,252, 4,992,445 and 5,001,139. Such patchesmay be constructed for continuous, pulsatile, or on demand delivery ofpharmaceutical agents.

For topical administration, all the formulations for topical ocularadministration used in the field of ophthalmology (e.g., eye drops,inserts, eye packs, impregnated contact lenses, pump delivery systems,dimethylsulfoxide (DMSO)-based solutions suspensions, liposomes, and eyeointment) and all the formulations for external use in the fields ofdermatology and otolaryngology (e.g., ointment, cream, gel, powder,salve, lotion, crystalline forms, foam, and spray) may be utilized as isknown in the art. Additionally all suitable formulations for topicaladministration to skin and mucus membranes of the nasal passages may beutilized to deliver the compounds of the invention. The pharmaceuticalcompositions of the present invention may be a liposomal formulation fortopical or oral administration, any of which are known in the art to besuitable for the purpose of this invention. For topical ocularadministration, the concentration of the drug may be adjusted, the pH ofthe solution buffered, and/or the isotonicity adjusted to be compatiblewith tear, nonirritating and well tolerated by the subject as is wellknown in the art.

Compositions may administered via oral delivery. Oral formulations cancomprise liquid formulations which are encapsulated or not. A liquidformulation may be an aqueous solution of the LFA-1 antagonist, and maycontain buffering agents and may or may not have preservatives included.Orally administered formulations such as tablets may optionally becoated or scored and may be formulated so as to provide sustained,delayed or controlled release of the active ingredient therein. Examplesof solid formulations may be as described in U.S. Pat. No. 5,424,289.Oral formulations can also have increased bioavailability, such asdescribed in U.S. Pat. No. 7,097,851, location and time dependent indelivery, such as described in U.S. Pat. No. 5,840,332, or delivered tospecific regions of the gastrointestinal system, for example, asdescribed in U.S. Pat. No. 5,849,327, where coating of an entericmaterial that remains intact until the dosage form reaches the lowergastrointestinal tract.

Formulations may use enteric coatings which are available for tabletsand capsules. Enteric coatings can remain intact in the stomach butrapidly dissolve when they arrive at the small intestine, thereafterreleasing the drug at sites downstream in the intestine (e.g., the ileumand colon), thus delivering a LFA-1 antagonist to the mucosa thereof.Enteric coatings are well known in the art and are discussed at, forexample, Remington's Pharmaceutical Sciences, Mack Publishing Co.,Easton, Pa.; and Polymers for Controlled Drug Delivery, Chapter 3, CRCPress, 1991. Some non-limiting examples of enteric coatings includecellulose acetate phthalate, polyvinyl acetate phthalate, methacrylicacid-methacrylic acid ester copolymers, carboxymethyl ethylcellulose,and hydroxypropyl methylcellulose acetate succinates. Alternatively, acontrolled release oral delivery vessel designed to release theformulations comprising a LFA-1 antagonist after a predetermined periodof time, and thus after the vessel has passed into the ileum or colon,can also be used to deliver the formulation of the present invention.Such vessels include, but are not limited to, the CHRONSET™ deliverydevice (ALZA Corporation, Palo Alto, Calif.) and the Pulsincap™ deliverydevice (R.P. Scherer Co.). Other coating agents may include, but not belimited to: sodium carboxymethyl cellulose, cellulose acetate phthalate,ethylcellulose, gelatin, pharmaceutical glaze, hydroxypropyl cellulose,hydroxypropyl methylcellulose, hydroxypropyl methyl cellulose phthalate,methylcellulose, polyethylene glycol, polyvinyl acetate phthalate,shellac, sucrose, titanium dioxide, camauba wax, microcrystalline wax,or mixtures thereof.

Controlled release oral formulations of the LFA-1 antagonist can also beformed wherein the LFA-1 antagonist is incorporated within abiocompatible and/or biodegradable matrix. The matrix can be hydrophilicor hydrophobic. Three main mechanisms exist by which an activeingredient can be released from a hydrophilic matrix: dissolution,erosion and diffusion. An active ingredient will be released by thedissolution mechanism when it is homogeneously dispersed in a matrixnetwork of a soluble polymer. The network will gradually dissolve in thegastrointestinal tract, thereby gradually releasing its load. The matrixpolymer can also gradually be eroded from the matrix surface, likewisereleasing the active ingredient in time. When an active ingredient isprocessed in a matrix made up of an insoluble polymer, it will bereleased by diffusion: the gastro-intestinal fluids penetrate theinsoluble, sponge-like matrix and diffuse back out loaded with drug.

The formulations of the present invention can contain the LFA-1antagonist as either a carboxylic acid or as a salt. The formulationscan include a polymer such as polylactic-glycoloic acid (PLGA),poly-(I)-lactic-glycolic-tartaric acid (P(I)LGT) (WO 01/12233),polyglycolic acid (U.S. Pat. No. 3,773,919), polylactic acid (U.S. Pat.No. 4,767,628), poly(M-caprolactone) and poly(alkylene oxide) (U.S.2003/0068384) to create a sustained release formulation, which may beliquid, gel, or a solid. Such formulations can be used to manufactureimplants that release a LFA-1 antagonist over a period of a few days, afew weeks or several months depending on the polymer, the particle sizeof the polymer, and the size of the implant (see, e.g., U.S. Pat. No.6,620,422). Other sustained release formulations and polymers for use inare described in EP 0 467 389 A2, WO 93/24150, U.S. Pat. No. 5,612,052,WO 97/40085, WO 03/075887, WO 01/01964A2, U.S. Pat. No. 5,922,356, WO94/155587, WO 02/074247A2, WO 98/25642, U.S. Pat. Nos. 5,968,895,6,180,608, U.S. 20030171296, U.S. 20020176841, U.S. Pat. Nos. 5,672,659,5,893,985, 5,134,122, 5,192,741, 5,192,741, 4,668,506, 4,713,244,5,445,832 U.S. Pat. Nos. 4,931,279, 5,980,945, WO 02/058672, WO 9726015,WO 97/04744, and US20020019446. In sustained release formulationsforming implants, microparticles of LFA-1 antagonist are combined withmicroparticles of polymer. One or more sustained release implants can beplaced in the large intestine, the small intestine or both. U.S. Pat.No. 6,011,011 and WO 94/06452 describe a sustained release formulationproviding either polyethylene glycols (i.e. PEG 300 and PEG 400) ortriacetin.

Another formulation which may both enhance bioavailability and providecontrolled release of the LFA-1 antagonist within the GI tract, is avariant of that described in WO 03/053401. Such a controlled releaseformulation includes a permeation enhancer, the LFA-1 antagonist, and acarrier that exhibits in-site gelling properties, such as a nonionicsurfactant. The formulation is delivered within the GI tract as a liquidhaving at least some affinity for the surface of the GI mucosalmembrane. Once released, the liquid formulation can spread across one ormore areas on the surface of the GI mucosal membrane, where the carrierof the formulation then transitions into a bioadhesive gel in-situ. As abioadhesive gel, the formulation of the present invention not onlyadheres to the mucosal membrane of the GI tract, but also reduces orminimizes dilution of both the permeation enhancer and the LFA-1antagonist included in the formulation by lumenal fluids and secretions.Bioavailability of the LFA-1 antagonist may be increased by presentingthe LFA-1 antagonist, together with a suitable permeation enhancer, atthe surface of the mucosal membrane of the GI tract at concentrationssufficient to increase absorption of the LFA-1 antagonist through the GImucosal membrane over a period of time.

Permeation enhancers suitable for use in a controlled formulation ofthis type include, but are not limited to, ethylene-diamine tetra-aceticacid (EDTA), bile salt permeation enhancers, such as sodiumdeoxycholate, sodium taurocholate, sodium deoxycholate, sodiumtaurodiliydrofusidate, sodium dodecylsulfate, sodium glycocholate,taurocholate, glycocholate, taurocheno-deoxycholate, taurodeoxycholate,deoxycholate, glycodeoxycholate, and ursodeoxycholate, fatty acidpermeation enhancers, such as sodium caprate, sodium laurate, sodiumcaprylate, capric acid, lauric acid, and caprylic acid, acyl carnitines,such as palmitoyl carnitine, stearoyl camitine, myristoyl carnitine, andlauroyl carnitine, and salicylates, such as sodium salicylate, 5-methoxysalicylate, and methyl salicylate. Permeation enhancers may act to openthe tight junctions formed between epithelial cells of the GI mucosalmembrane, and thereby allow diffusion of the LFA-1 antagonist into theintestinal mucosa (i. e., pericellular absorption. Though the amount ofpermeation enhancer included in the formulation of the present inventionmay range from about 10 wt % to about 40 wt %, the nature and preciseamount of permeation enhancer included in the formulation of the presentinvention will vary depending on, for example, the LFA-1 antagonist tobe delivered, the nature of the permeation enhancer itself, and the doseof formulation to be administered. The amount of permeation enhancerincluded in the formulation should be sufficient to maintain aneffective concentration of permeation enhancer (i.e., a concentrationabove the critical concentration for the permeation enhancer used) at ornear the surface of the GI mucosal membrane over a period of timesufficient to increase the bioavailability of the LFA-1 antagonist.Where possible, the permeation enhancer can be chosen such that thepermeation enhancer not only facilitates absorption of the LFA-1antagonist, but also resists dilution by lumenal fluids or secretions.Permeation enhancers may also be used in formulations of the inventionwhich are not controlled release formulations.

The carrier of a controlled release formulation containing a permeationenhancer, the LFA-1 antagonist, and the carrier exhibiting in-sitegelling properties will permit a transition from a relativelynon-adhesive, low viscosity liquid to a relatively viscous, bioadhesivegel after the formulation has been delivered within the GI tract of asubject. The carrier is chosen such that the transition from arelatively non-adhesive, low viscosity liquid to a relatively viscous,bioadhesive gel occurs after the formulation has been released withinthe GI tract and had some opportunity to arrive at the surface of the GImucosal membrane. Hence, the carrier of the formulation of the presentinvention enables the in-situ transition of the formulation from aliquid to a bioadhesive gel. Due to its high viscosity and bioadhesiveproperties, the gel formed by the formulation of the present inventionholds the permeation enhancer and the LFA-1 antagonist together at thesurface of the GI mucosal membrane and protects both such componentsfrom dilution and enzymatic degradation over a period of time. Suitablecarriers include non-ionic surfactants that transition from a relativelynon-adhesive, low viscosity liquid to a relatively viscous, bioadhesiveliquid crystal state as they absorb water. Specific examples ofnon-ionic surfactants that may be used as the carrier in the formulationof the present invention include, but are not limited to, Cremophor (e.g., Cremophor EL and Cremophor RH), Incordas 30, polyoxyethylene 5castor oil, polyethylene 9 castor oil, polyethylene 15 castor oil,d-a-tocopheryl polyethylene glycol succinate (TPGS), monoglycerides,such as myverol, aliphatic alcohol based nonionic surfactants, such asoleth-3, oleth-5, polyoxyl 10 oleyl ether, oleth-20, steareth-2,stearteth-10, steareth-20, ceteareth-20, polyoxyl 20 cetostearyl ether,PPG-5 ceteth-20, and PEG-6 capryl/capric triglyceride, Pluronic® andtetronic block copolymer non-ionic surfactants, such as Pluronic® L10,L31, L35, L42, L43, L44, L62, L61, L63, L72, L81, L101, L121, and L122,polyoxylene sorbitan fatty acid esters, such as Tween 20, Tween 40,Tween 60, Tween 65, Tween 80, Tween 81, and Tween 85, and ethoxylatedglycerides, such as PEG 20 almond glycerides, PEG-60 almond glycerides,PEG-20 corn glycerides, and PEG-60 corn ARC 2921 PCT 11 glycerides. Thecarrier may be present in about 35 wt % to about 88 wt % of theformulation.

As water is added to the controlled release formulation having anon-ionic surfactant as the carrier, the initial viscosity of theformulation will increase. However, as water content increases, theincrease in viscosity of nonionic surfactants tends to be non-linear.Often, as the water content of a nonionic surfactant exceeds a certainthreshold, the viscosity of the nonionic surfactant increases rapidly asthe nonionic surfactant transitions to its gelling state. If arelatively quick conversion is desired, a formulation including anonionic surfactant may be provided more water, thereby placing theformulation closer to the water content threshold at which theformulation will rapidly convert to a bioadhesive gel. In contrast, if arelatively slow conversion is desired, the formulation may include lesswater or no water, thereby placing the formulation farther from thegelling threshold.

Additionally, the controlled release formulation containing a permeationenhancer, the LFA-1 antagonist, and the carrier exhibiting in-sitegelling properties may also include a viscosity reducing agent thatreduces the initial viscosity of the formulation. Reducing the initialviscosity of the formulation may further facilitate spreading of theformulation of the present invention across one or more areas of the GImucosal membrane after the formulation is delivered within the GI tractbut before the formulation transitions into a bioadhesive gel.

Exemplary viscosity reducing agents that may be used include, but arenot limited to, polyoxyethylene 5 castor oil polyoxyethylene 9 castoroil, labratil, labrasol, capmul GMO (glyceryl mono oleate), capmul MCM(medium chain mono- and diglyceride), capmul MCM C8 (glyceryl monocaprylate), capmul MCM C10 (glyceryl mono caprate), capmul GMS-50(glyceryl mono stearate), caplex 100 (propylene glycol didecanoate),caplex 200 (propylene glycol dicaprylate/dicaprate), caplex 800(propylene glycol di 2-ethyl hexanoate), captex 300 (glyceryltricapryl/caprate), captex 1000 (glyceryl tricaprate), captex 822(glyceryl triandecanoate), captex 350 (glyceryltricaprylate/caprate/laurate), caplex 810 (glyceryltricaprylate/caprate/linoleate), capmul PG8 (propylene mono caprylate),propylene glycol, and propylene glycol laurate (PGL). Where a viscosityreducing agent is included, the viscosity reducing agent may be presentin amounts up to about 10 wt % of the formulation.

Further, the dosage form of the controlled release formulationcontaining a permeation enhancer, the LFA-1 antagonist, and the carrierexhibiting in-site gelling properties may include a hard or soft gelatincapsule. In some embodiments, the dosage form is designed, such as byuse of enteric coatings, to delay release of the formulation until thedosage form has passed through the stomach and at least entered thesmall intestine.

Additional controlled release formulations are described in WO 02/38129,EP 326 151, U.S. Pat. No. 5,236,704, WO 02/30398, WO 98/13029; U.S.20030064105, U.S. 20030138488A1, U.S. 20030216307A1, U.S. Pat. No.6,667,060, WO 01/49249, WO 01/49311, WO 01/49249, WO 01/49311, and U.S.Pat. No. 5,877,224. An example of a solid controlled release formulationmay include hydrophilic polymers such as starch, cellulosic polymers,polyacrylic acids, or polymethacrylic acids to entrap the LFA-1antagonist; cyclodextrins such as alpha, beta, or gamma cyclodextrinsand further including substituted cyclodextrins such as sulfobutylcyclodextrins or hydroxypropyl beta cyclodextrin, which complex with theLFA-1 antagonist and act to provide a more regulated release of theLFA-1 antagonist from the solid controlled release formulation.

The oral administration formulations may utilize gastroretentiveformulations to enhance absorption from the gastrointestinal (GI) tract.A formulation which is retained in the stomach for several hours mayrelease compounds of the invention to provide a sustained release in theupper gastrointestinal region that may be desirable in some embodimentsof the invention. Disclosure of such gastro-retentive formulations arefound in Klausner, E. A.; Lavy, E.; Barta, M.; Cserepes, E.; Friedman,M.; Hoffman, A. 2003 “Novel gastroretentive dosage forms: evaluation ofgastroretentivity and its effect on levodopa in humans.” Pharm. Res. 20,1466-73, Hoffman, A.; Stepensky, D.; Lavy, E.; Eyal, S. Klausner, E.;Friedman, M. 2004 “Pharmacokinetic and pharmacodynamic aspects ofgastroretentive dosage forms” Int. J. Pharm. 11, 141-53, Streubel, A.;Siepmann, J.; Bodmeier, R.; 2006 “Gastroretentive drug delivery systems”Expert Opin. Drug Deliver. 3, 217-3, and Chavanpatil, M. D.; Jain, P.;Chaudhari, S.; Shear, R.; Vavia, P. R. “Novel sustained release,swellable and bioadhesive gastroretentive drug delivery system forolfoxacin” Int. J. Pharm. 2006 epub March 24. Expandable, floating andbioadhesive techniques may be utilized to maximize the extent and/orduration of absorption of the compounds of the invention. Materials suchas cross povidone, pysllium husk, chitosan, cellulosic polymers, amongstother materials, can be selected and combined to vary the buoyancy lagtime, duration of buoyancy, dimensional stability, drug content and drugrelease profile. Variation of the physical characteristics of theformulation can also used to vary these parameters of drug delivery. Forexample, a biodegradable membrane may be included as part of agastroretentive formulation, which membrane is buoyant in the stomachand is exposed to the gastric environment only over a predetermined timeperiod. This membrane may be formed of materials that only release LFA-1antagonist after this initial exposure period, thus, in combination withan immediately releasing portion of the gastroretentive formulation,providing LFA-1 antagonist over a much extended period of time relativeto a formulation comprising only immediate release compositions.

An aerosol suspension of respirable particles may be solid or liquid,with suitably sized particles, as is known in the art to be effectivefor absorption. Compositions for inhalation or insufflation includesolutions and suspensions in pharmaceutically acceptable, aqueous ororganic solvents, or mixtures thereof, and powders. The liquid or solidcompositions may contain suitable pharmaceutically acceptable excipientsas described supra. In some embodiments, the compositions areadministered by the oral or nasal respiratory route for local orsystemic effect. Compositions in pharmaceutically acceptable solventsmay be nebulized by use of inert gases. Nebulized solutions may beinhaled directly from the nebulizing device or the nebulizing device maybe attached to a face mask tent, or intermittent positive pressurebreathing machine. Alternatively a pressurized metered dose device maybe used to deliver the drug, or dry powder inhaler devices. Solution,suspension, or powder compositions may be administered, topically,orally or nasally, from devices that deliver the formulation in anappropriate manner.

Eye drops may be prepared by dissolving the amorphous form or any of thecrystalline forms of the compound of Formula I, or a combinationthereof, in a sterile aqueous solution such as physiological saline,buffering solution, etc., or by combining powder compositions to bedissolved before use. Other vehicles may be chosen, as is known in theart, including but not limited to: balance salt solution, salinesolution, water soluble polyethers such as polyethyene glycol,polyvinyls, such as polyvinyl alcohol and povidone, cellulosederivatives such as methylcellulose and hydroxypropyl methylcellulose,petroleum derivatives such as mineral oil and white petrolatum, animalfats such as lanolin, polymers of acrylic acid such ascarboxypolymethylene gel, vegetable fats such as peanut oil andpolysaccharides such as dextrans, and glycosaminoglycans such as sodiumhyaluronate. If desired, additives ordinarily used in the eye drops canbe added. Such additives include isotonizing agents (e.g., sodiumchloride, etc.), buffer agent (e.g., boric acid, sodium monohydrogenphosphate, sodium dihydrogen phosphate, etc.), preservatives (e.g.,benzalkonium chloride, benzethonium chloride, chlorobutanol, etc.),thickeners (e.g., saccharide such as lactose, mannitol, maltose, etc.;e.g., hyaluronic acid or its salt such as sodium hyaluronate, potassiumhyaluronate, etc.; e.g., mucopolysaccharide such as chondroitin sulfate,etc.; e.g., sodium polyacrylate, carboxyvinyl polymer, crosslinkedpolyacrylate, polyvinyl alcohol, polyvinyl pyrrolidone, methylcellulose, hydroxy propyl methylcellulose, hydroxyethyl cellulose,carboxymethyl cellulose, hydroxy propyl cellulose or other agents knownto those skilled in the art).

In some embodiments of the invention, addition of sodium bicarbonate ismade to the amorphous form or any of crystalline forms A, B, C, D, or E,or a mixture thereof of the compound of Formula I to convert it to asodium salt. In some embodiments of the invention, the amorphous form orany of the crystalline forms A, B, C, D, or E are formulated as theirsodium, potassium, lithium, magnesium, zinc, or calcium salts.

The solubility of the components of the present compositions may beenhanced by a surfactant or other appropriate co-solvent in thecomposition. Such cosolvents include polysorbate 20, 60, and 80,Pluronic F68, F-84 and P-103, cyclodextrin, or other agents known tothose skilled in the art. Such co-solvents may be employed at a level offrom about 0.01% to 2% by weight.

The composition of the invention can be formulated as a sterile unitdose type containing no preservatives.

The compositions of the invention may be formulated as a sterile unitdose type containing preservatives. Suitable preservatives includebenzalkonium chloride, thimerosal, chlorobutanol, methyl paraben, ethylparaben, propyl paraben, phenylethyl alcohol, imidazolidinyl urea,diazolidinyl urea, phenoxyethanol, edetate disodium, sorbic acid, OnamerM, purite, peroxides, perborates, and other agents known to one of skillin the art. The preservatives may be employed at a level from about0.004% to about 0.02% W/W.

The compositions of the invention may be packaged in multidose form.Preservatives may be preferred to prevent microbial contamination duringuse. Suitable preservatives include: benzalkonium chloride, thimerosal,chlorobutanol, methyl paraben, ethyl paraben, propyl paraben,phenylethyl alcohol, imidazolidinyl urea, diazolidinyl urea,phenoxyethanol, edetate disodium, sorbic acid, Onamer M, or other agentsknown to those skilled in the art. In the prior art ophthalmic products,such preservatives may be employed at a level of from 0.004% to 0.02%.

In some of the compositions of the present application, methyl parabenand propyl paraben are used in combination.

In the compositions of the present application the preservativebenzalkonium chloride, may be employed at a level of from about 0.001%to less than about 0.01%, e.g. from about 0.001% to about 0.008%, orabout 0.005% by weight. It has been found that a concentration ofbenzalkonium chloride of about 0.005% may be sufficient to preserve thecompositions of the present invention from microbial attack.

The formulations of the invention can further include otherpharmacological active ingredients as far as they do not contradict thepurpose of the present invention. In a combination of plural activeingredients, their respective contents may be suitably increased ordecreased in consideration of their effects and safety.

Kits

The invention also provides kits. The kits include a compound of theinvention in suitable packaging, and written material that can includeinstructions for use, discussion of clinical studies, listing of sideeffects, and the like. The kit may further contain another therapeuticagent that is co-administered with the amorphous form or any of thecrystalline Forms of the compound of Formula I or a combination thereof.In some embodiments, the therapeutic agent and the amorphous form or anyof the crystalline Forms of the compound of Formula I or a combinationthereof are provided as separate compositions in separate containerswithin the kit. In some embodiments, the therapeutic agent and theamorphous form or any of the crystalline forms of the compound ofFormula I or a combination thereof are provided as a single compositionwithin a container in the kit. Suitable packaging and additionalarticles for use (e.g., measuring cup for liquid preparations, foilwrapping to minimize exposure to air, dispensers, and the like) areknown in the art and may be included in the kit.

EXAMPLES Example 1: Human T-Cell Adhesion Assay

The T-cell adhesion assay was performed using the human T-lymphoid cellline HuT 78 (ATCC TIB-161). Goat anti-HuIgG(Fc) was diluted to 2 μg/mlin PBS and 96-well plates were coated with 50 μl/well at 37° C. for 1 h.Plates were washed with PBS and blocked for 1 h at room temperature with1% BSA in PBS. 5 domain ICAM-Ig was diluted to 100 ng/ml in PBS and 50μl/well was added to the plates O/N at 4° C. HuT 78 cells werecentrifuged at 100 g and the cell pellet was treated with 5 mM EDTA for˜5′ at 37° C. in a 5% CO₂ incubator. Cells were washed in 0.14 M NaCl,0.02 M Hepes, 0.2% glucose and 0.1 mM MnCl₂ (assay buffer) andcentrifuged. The cells were resuspended in assay buffer to 3.0×10⁶ c/ml.Inhibitors were diluted in assay buffer to a 2× final concentration andpre-incubated with HuT78 cells for 30′ at room temperature. 100 μl/wellof cells and inhibitors were added to the plates and incubated at roomtemperature for 1 h. 100 μl/well PBS was added and the plates weresealed and centrifuged inverted at 100 g for 5′. Unattached cells wereflicked out of the plate and excess PBS was blotted on a paper towel. 60μl/well p-nitrophenyl n-acetyl-o-D-glucosaminide (0.257 g to 100 mlcitrate buffer) was added to the plate and incubated for 1.5 h at 37° C.The enzyme reaction was stopped with 90 μl/well 50 mM glycine/5 mM EDTAand read on a platereader at 405 nM. HUT 78 cell adhesion to 5dICAM-Igwas measured using the p-nitrophenyl n-acetyl-β-D-glucosaminide methodof Landegren, U. (1984). J. Immunol. Methods 57, 379-388. The compoundof Formula was shown to be a potent inhibitor of T-cell adhesion with anEC 50 of less than 50 nM.

Example 2: LFA-1:ICAM-1 Receptor Binding Assay Using Forward FormatAssay

Competitive inhibition of the LFA-1:ICAM-1 interaction is quantitated byadding known amounts of inhibitors.

Purified full length recombinant human LFA-1 protein is diluted to 2.5μg/ml in 0.02 M Hepes, 0.15M NaCl, and 1 mM MnCl₂ and 96-well plates (50μl/well) are coated overnight at 4° C. The plates are washed with washbuffer (0.05% Tween in PBS) and blocked for 1 h at room temperature with1% BSA in 0.02M Hepes, 0.15 M NaCl, and 1 mM MnCl₂. Plates are washed.50 μl/well inhibitors, appropriately diluted in assay buffer (0.5% BSAin 0.02M Hepes, 0.15M NaCl, and 1 mM MnCl₂), are added to a 2× finalconcentration and incubated for 1 h at room temperature. 50 μl/well ofpurified recombinant human 5 domain ICAM-Ig, diluted to 50 ng/ml inassay buffer, is added and incubated 2 h at room temperature. Plates arewashed and bound ICAM-Ig is detected with Goat anti-HuIgG(Fc)-HRP for 1h at room temperature. Plates are washed and developed with 100 μl/wellTMB substrate for 10-30′ at room temperature. Colorimetric developmentis stopped with 100 μl/well 1M H₂PO₄ and read at 450 nM on aplatereader. The compound of Formula I is expected to demonstrate potentcompetitive inhibition of the interaction between LFA-1 and ICAM-1.

Example 3: In-Vitro Inhibition of Antigen Stimulated Release ofCytokines from Human Peripheral Blood Monocytes (PBMC)

One form of the LFA-1 antagonist, the compound of Formula I, wasevaluated for its ability to inhibit release of inflammatory cytokines,in human mononuclear cells (PBMC) stimulated with staphylococcalenterotoxin B (SEB). Stock solutions of the compound of Formula I,Rebamipide (a mucosal protective agent), and Cyclosporin A (CsA) wereprepared in culture media and dilutions were prepared by addition ofculture media to achieve the desired concentration. Negative controlswere prepared without SEB stimulation. SEB stimulation with vehicle(0.25% DMSO/media) was used as the positive control. The compound ofFormula I was shown to have EC50 of 150 nM or less.

Human PBMC, frozen in cryopreservation media were thawed, washed withRPMI culture media containing 10% FBS in growth media and seeded onto a96 well plate at 20,000 cells/well containing 180 μl culture media.Cells were incubated in the presence of the compound of Formula I,Rebamipide or CsA at 37° C. for 1 hour prior to stimulation with SEB.SEB was added at 1 ng/ml and cell supernatants were harvested at 6, 16,and 48 hours. Cytokine levels in the assay supernatants were determinedusing a Luminex multiplex assay.

The compound of Formula I demonstrated potent inhibition of the releaseof inflammatory cytokines, particularly the T-cell regulating cytokines,IL-2 and IL-4, with increasing dose. The results are shown in Tables 7,8, and 9. The pattern of cytokine release inhibited by more than 50%with the compound of Formula I is similar to that seen in comparisonwith CsA. The exceptions to this similarity include IL-3, 11-6, andIL-12p40.

TABLE 7 EC50 Concentrations for Inhibition of IL-2, IFNγ, MIP-1α, andTNF-α. EC50 μM Cytokine Release IL-2 IFNγ MIP-1α TNF-α Compound of0.0018 0.0016 0.020 0.076 Formula I Rebamipide >1000 >1000 >1000 >1000Cyclosporine A 0.00094 0.00050 0.0011 0.00049

TABLE 8 EC50 Concentrations for Inhibition of IL-4, IL-10, IP-10, GM-CSFand MCP-1. EC50 μM Cytokine Release IL-4 IL-10 IP-10 GM-CSF MCP-1Compound of 0.143 0.147 1.158 0.545 0.0050 Formula IRebamipide >1000 >1000 >1000 >1000 >1000 Cyclosporine A 0.0063 0.02920.167 0.0202 0.0926

TABLE 9 EC50 Concentrations for Inhibition of IL-1α, IL-1β, IL-3, IL-5,IL-6, IL-12p40, and IL-13. EC50 μM Cytokine Release IL-1α IL-1β IL-3IL-5 IL-6 IL-12p40 IL-13 Compound of 0.24 0.36 52.23 0.11 43.51 >10000.36 Formula I Rebamipide >1000 >1000 >1000 >1000 >1000 >1000 >1000Cyclosporine A 0.002 0.003 0.002 0.073 0.001 0.002 0.074

Example 4: Formulations of LFA-1 Antagonist

The compound of Formula I was formulated in several compositions foradministration as gels, lotions, ointments, and solutions, foradministration by varying routes, including but not limited to topical,via instillation, aerosol, transdermal patch, via insert, or oraladministration.

TABLE 10 Gel Formulations 1 and 2 of the Compound of Formula I.Formulation 1 Formulation 2 (% w/w) (% w/w) 1% Form A of the 1% Form Aof the compound of compound of Formula I Formula I 15% Dimethyl 15%Dimethyl Isosorbide Isosorbide 25% Transcutol 25% Transcutol 12%Hexylene glycol 12% Hexylene glycol 5% Propylene Glycol 5% PropyleneGlycol 0.15% Methylparaben 0.15% Methylparaben 0.05% Propylparaben 0.05%Propylparaben 0.01% EDTA 0.01% EDTA 0.5% Penmulen TR-1 1% HydroxyethylCellulose q.s. pH 6.0 25% q.s. pH 4.5 25% Trolamine Trolamine q.s. 100Water q.s. 100 Water

TABLE 11 Lotion Formulations 3 and 4 of the Compound of Formula I.Formulation 3 (% w/w) Formulation 4 (% w/w) 1% Form A 1% Form A 13%Dimethyl Isosorbide 13% Dimethyl Isosorbide 20% Transcutol 20%Transcutol 10% Hexylene glycol 10% Hexylene glycol 4% Propylene Glycol4% Propylene Glycol 0.15% Methylparaben 0.15% Methylparaben 0.05%Propylparaben 0.05% Propylparaben 0.01% EDTA 0.01% EDTA 0.5% CarbopolUltrez 10 0.3% Carbopol Ultrez 10 0.2% Penmulen TR-1 0.2% Penmulen TR-13% Isopropyl Myristate 2% Cetyl Alcohol 5% Olelyl Alcohol 5.5% LightMineral Oil 5% White Petrolatum 5% Oleic Acid 0.02% Butylated 0.02%Butylated Hydroxytoluene Hydroxytoluene q.s. pH 6.0 25% Trolamine q.s.pH 6.0 25% Trolamine q.s. 100 Water q.s. 100 Water

TABLE 12 Ointment Formulations 5 and 6 of the Compound of Formula I.Formulation 5 (% w/w) Formulation 6 (% w/w) 1% Form A 1% Form A 15% PEG400 10% Dimethyl Isosorbide 0.02% Butylated 0.02% ButylatedHydroxytoluene Hydroxytoluene 2% Span 80 2% Span 80 10% White Wax 10%White Wax 71.98% White Petrolatum 76.98% White Petrolatum

TABLE 13 Solution Formulations 7, 8, and 9 of the Compound of Formula I.Formulation 7 Formulation 8 Formulation 9 (% w/w) (% w/w) (% w/w) 1%Form A 1% Form A 1% Form A 15% Dimethyl 15% Dimethyl Isosorbide 99%Dimethyl Isosorbide Sulfoxide 25% Transcutol 25% Transcutol 12% Hexyleneglycol 12% Hexylene glycol 5% Propylene Glycol 5% Propylene Glycol q.s.pH 4.5 25% q.s. pH 6.0 25% Trolamine Trolamine q.s. 100 Water q.s. 100Water

TABLE 14 Solution Formulations 10, 11, 12, 13 and 14 of the Compound ofFormula I. Formulation Formulation Formulation Formulation FormulationW/W % 10 11 12 13 14 Form A  0.1%  0.3%   1%   3%   5% SodiumBicarbonate 0.015% 0.046% 0.15% 0.46% 0.77% 0.1% EDTA 0.12% SodiumPhosphate, Monobasic 0.4% Methylparaben 0.02% Propylparaben q.s.Osmolality 270, Sodium Chloride q.s. pH 7.0 1% Sodium Hydroxide q.s. pH7.0 1% HCl q.s. Water

TABLE 15 Solution Formulation 15 of the Compound of Formula I.Formulation 15 1 ml of a solution of the compound of Formula I 10% W/Win water, plus 0.158 mmol sodium bicarbonate 9 ml PBS

The compound of Formula I can be supplied as a sterile, clear, colorlessliquid solution containing 0.1%, 1.0%, and 5.0% (w/w) ActivePharmaceutical Ingredient (API) concentrations (pH 7.0). Each mL of a 1%solution contains 10 mg of the active ingredient. In addition to thecompound of Formula I, other components of a drug product solution,their functions, and their compendial grade can include propylparaben(preservative; National Formulary (NF)), methylparaben (preservative,NF), EDTA (antioxidant, United States Pharmacopeia (USP)), sodiumbicarbonate (buffering agent, USP), monobasic sodium phosphate(buffering agent, USP), dibasic sodium phosphate (buffering agent, USP),and sterile water (diluent, USP). All excipients can be of compendialgrade and of non-human or non-animal origin.

Formulated drug product solution can be packaged under asepticconditions into sterile 7.0 mL High Density Polyethylene (HDPE) bottlesequipped with a dropper tip that delivers an approximate per drop volumeof 0.35 μL and a protective cap. The dropper bottle can have a 40 μLtip. Unpreserved study drug (no methyl or propylparabens in theformulation) can be provided in 0.5 mL unit dose Low DensityPolyethylene (LDPE) containers manufactured using a blow fill sealprocess and stored in aluminum foil pouches.

Drug solutions can be stored refrigerated (2-8° C.). The stability ofthe drug at 5° C. and 25° C. can be out to 9 months or longer.

Example 5: In-Vitro Percutaneous Absorption of the Compound of Formula IFollowing Topical Application

Bioavailability following topical application in-vivo was assessed usingin-vito percutaneous absorption test methods, using procedures adaptedfrom Skelly et al., Pharmaceutical Research 1987 4(3): 265-276, “FDA andAAPS Report of the Workshop on Principles and Practices of In-VitroPercutaneous Penetration Studies: Relevance to Bioavailability andBioequivalence”.

Formulations 1-9 were applied to dermatomed human skin tissue excisedfrom a single donor in a single clinically relevant dose of 5 mg/cm²,which is equivalent to a 30-35 μg dose. The thickness of the tissueranges form 0.023 to 0.039 inches (0.584 to 0.991 mm) with amean+/−standard deviation in thickness of 0.030+/−0.004 inches(0.773+/−0.111 mm) and a coefficient of variation of 14.4%. The tissuesamples were mounted in Bronaugh flow-through diffusion cells. The cellswere maintained at a constant temperature of 32° C. using recirculatingwater baths. The cells have a nominal diffusion area of 0.64 cm². PBS,at pH 7.4, with 0.1% sodium azide and 4% Bovine Serum Albumin was usedas the receptor phase below the mounted tissue. Fresh receptor phase wascontinuously pumped under the tissue at a flow rate of nominally 1.0ml/hr and collected in 6 hour intervals. The receptor phases werecollected for analysis.

The tissue samples were exposed to Formulations 1-9 for 24 hours. Theexcess formulation residing on the strateum corneum at that timepointwas removed by tape-stripping with CuDerm D-Squame stripping discs. Thetape strips were discarded. The epidermis and dermis were separated byblunt dissection. Epidermis, dermis and receptor phase were analyzed forcontent of the compound of Formula I. The results are represented inTable 16.

Tissue permeation levels (the receptor phase) of the compound of FormulaI for all formulations except for Formulation 9, which contained 99%DMSO, were below the limits of quantitation, which was 0.54 ng/ml (whichis equivalent to 0.013% of the applied dose). Formulation 9, incontrast, provided 1.4% of the applied dose, permeating through all thelayers of the skin tissue over the exposure period of 24 hours.

Epidermal deposition of the compound of Formula I over the 24 hourexposure period was very high and consistent with a large percentage ofthe applied dose being retained in the upper layers of the epidermis.The levels reported in Table 10 were obtained from small volume samples,which could not be re-assayed, and thus are considered underestimates ofthe amount of drug present in the epidermis.

Analytical data for the dermis fell within the linearity rangeestablished for the compound of Formula I, and are quantitative. Dermaldeposition of the compound of Formula I following a 24 hour exposureranged from 0.66% (Formulation 6, 0.258 μg/cm²) to 4.4% (Formulation 7,343 μg/cm²) of the applied dose. The concentration of the compound ofFormula I (633.5 g/mole) in the dermis is thereby calculated as 6.7 μM(Formulation 6) or greater (i.e., Formulation 7 provides a concentrationin the dermis of 54.1 μM) for Formulations 1 to 9 in the dermis. Theseconcentrations are well above the in-vitro EC50 concentration for halfmaximal effect in inhibiting release of inflammatory cytokines by thecompound of Formula I, as shown in Example 3. These results aretherefore predictive for the ability of a variety of formulations, whichincorporate 1% W/W of the compound of Formula I, to providetherapeutically effective levels of in-vivo inhibition of cytokinerelease.

TABLE 16 Cumulative Receptor Phase and Tissue Levels of the Compound ofFormula I After 24 Hours of Topical Exposure. Receptor Phase Content at24 hours Epidermis Dermis % Dose % Dose % Dose Formulation # μg/cm²Applied μg/cm² Applied μg/cm² μg/ml Applied 1 Mean <Limit ofQuantitation 3.93 7.48 1.14 18.8 2.15 SD¹ 2.92 5.50 0.91 14.9 1.73 % CV²74 74 80 80 80 2 Mean <Limit of Quantitation 6.03 11.9 0.750 12.3 1.49SD 2.56 5.1 0.304 5.0 0.63 % CV 43 42 40 40 42 3 Mean <Limit ofQuantitation 6.03 12.1 1.40 23.0 2.74 SD 2.97 6.4 0.27 4.4 0.47 % CV 4953 19 19 17 4 Mean <Limit of Quantitation 7.92 17.0 0.975 16.0 2.10 SD3.41 7.2 0.350 5.8 0.75 % CV 43 42 36 36 36 5 Mean <Limit ofQuantitation 5.71 14.6 0.670 11.0 1.71 SD 1.73 4.2 0.351 5.8 0.87 % CV30 29 52 52 51 6 Mean <Limit of Quantitation 6.47 16.8 0.258 4.25 0.657SD 1.07 2.7 0.158 2.6 0.394 % CV 17 16 61 61 60 7 Mean <Limit ofQuantitation 7.22 15.0 2.08 34.3 4.35 SD 2.15 4.5 0.84 13.7 1.83 % CV 3030 40 40 42 8 Mean <Limit of Quantitation 8.58 18.0 1.48 24.3 3.09 SD3.53 7.7 0.99 16.2 2.07 % CV 41 43 67 67 67 9 Mean 0.660 1.43 5.78 13.21.19 19.6 2.63 SD 0.253 0.49 3.18 8.3 0.49 8.1 1.15 % CV 38 34 55 63 4141 44 ¹Standard Deviation ²Percent Coefficient of Variation.

Example 6: Pharmacological Activity of the Compound of Formula I forTreatment of Keratoconjunctivitis Sicca (KCS)

Dogs were enrolled in this study if the following criteria were met:more than one year of age, a Schimer tear test (STT) of less than 10 mmwetting per minute, bilateral involvement, and at least one of thefollowing clinical signs: blepharospasm, conjunctivial hyperemia,exposure keratopathy (irregular surface), corneal pigmentation, cornealneovascularization or ropey mucopurulent discharge, no congenital KCS,no traumatic KCS, toxic KCS, and no facial nerve paralysis. If dogs hadbeen treated with topical CsA or tacrolimus in the previous six months,they were not enrolled.

The dogs were administered one 35 μl drop of the compound of Formula I,1% solution (Formulation 15, 0.35 mg/eye), in each affected eye threetimes daily, with approximately 4 hours (+1 hour) between the dailydoses for 12 weeks. CsA will be administered for a further four weeks byadministering commercially available 0.2% ointment three times a day,after the compound of Formula I is discontinued at twelve weeks.

Animals were subjected to an ocular examination once during the initialvisit and during five visits over sixteen weeks of the study (Weeks, 2,4, 8, 12 and 16). The last OE was approximately four weeks after thelast dose of the compound of Formula I and after one month of CsAtreatment. The adnexa and anterior portions of both eyes were examinedusing an indirect opthalmoscope. The eyes were dilated with a mydriaticwhen applicable, to allow evaluation of the lens and fundus, includingthe retina. An evaluation using a modified McDonald-Shaddock scoringsystem was performed in conjunction with the slitlamp ocularexaminations at each interval.

Tears were measured using STT strips during the initial visit and eachof the five follow-up visits on Weeks 2, 4, 8, 12 and 16. One strip ofSTT paper was used for each eye for each interval. At each collectioninterval, the STT paper was folded and placed in the inferior cul de sacfor sixty seconds. The length, in mm, of wetting below the notch of thepaper was recorded.

Fluorescein and rose bengal staining was performed at the each of theinitial and follow up examinations. Intraocular pressure measurements(IOPs) were performed using a Tono-Pet Vets in conjunction with each ofthe OEs. Digital ocular images were taken before and after staining(with fluorescein and rose bengal) during each of the OEs.

Conjunctival biopsies were taken at the initial (pretreatment) visit andthe Week 12 visit. The second biopsy was taken more lateral (approx. 1mm) to the initial biopsy. Following appropriate preparation a smallconjunctival biopsy was taken from the ventral fornix of each eye.

Seven dogs completed the study; for two dogs, only one eye was studied.The results are shown in Tables 17 and 18. Overall, a 3.3 mm averageimprovement in OD (right eye) STT and 4.5 mm in OS (left eye) STT wasobserved during the treatment period with the compound of Formula I.Results for all 12 eyes show an average of 4 mm improvement. AMaximum-Minimum analysis was performed using the maximal change in STTvalues for each eye in each dog over weeks 1-12, as shown in Table 19.This calculation yields a total maximal change in STT for total of eyesof 72 mm, which upon division by 12 (number of KCS eyes in theanalysis), yields a 6.0 mm average improvement. Other clinical signsimproved in some dogs, such as a decrease in mucopurulent discharge orconjunctival erythema. Histopathological evaluation of biopsies takenbefore and after the compound of Formula I revealed an attenuation oflymphocyte accumulation. FIG. 27 illustrates this phenomenon in samplestaken from dog #1. No significant additional benefit was seen from foursubsequent weeks of CsA administration.

TABLE 17 Schirmer Tear Test Results (OD). Dog ID Week 1 Week 2 Week 4Week 8 Week 12 Week 16 1 15 18 12 16 13 12 2 0 2 0 8 8  8 3 6 11 5 7 7 8 4 5 11 10 7 13  8 5 8 11 10 11 9  22** 6 8 10 15 17 16 18 7 6 2 2 1 012 Mean* 5.5 7.8 7.0 8.5 8.8   11.7 *Dog #1 not included in mean orMaximum-Minimum analysis for OD as there is no KCS in that eye for thatanimal. **Data for Dog #5 is anomalous for this day, and is not includedin the mean or Maximum-Minimum analysis.

TABLE 18 Schirmer Tear Test Results (OS). Dog ID Week 1 Week 2 Week 4Week 8 Week 12 Week 16 1 0 0 0 0 3 3 2 0 0 0 2 7 5 3 9 14 7 17 15 16 4 03 5 6 4 7 5 7 8 14 8 8 19 6 9 4 14 8 8 17 7 18 NA NA 19 18 18 Mean* 4.24.8 6.7 6.5 8.7 11.0 *Dog #7 not included in mean or Maximum-Minimumanalysis for OS as there is no KCS in that eye for that animal.

TABLE 19 Maximum-Minimum Analysis for Weeks 1-12 of the compound ofFormula I Administration. OD OS Total OD plus Total OS: NA 3 72 8 7 5 10Grand Total/Number of 8 6 Eligible Eyes: 3 7 6.0 mm Average 8 11Improvement −4   NA Total = 28 Total = 44

FIG. 28 illustrates the mean change in Schirmer test score at weeks 2,4, 8, and 12. Significant improvement in Schirmer test scores overpretreatment was observed in week 12.

FIG. 29 illustrates the percentage of eyes with a Schirmer test score ofgreater than 10 mm at 2, 4, 8, and 12-weeks with 1% the compound ofFormula I (TID). The compound of Formula I canine KCS study resultsexceeded human CsA data. The basis of restasis approval was animprovement of Schirmer test score to greater than 10 m. Restasistreatment resulted in 15% of eyes with Schirmer test score greater than10 mm.

FIG. 30 illustrates the percentage of eyes with a greater than 4 mmimprovement in Schirmer test score at 2, 4, 12, 16, and 26 weeks forsubjects treated with 1% the compound of Formula I (tid) or 2% CsA (bid)(using historic CsA data; Morgan et al., J. Am. Vet. Med. Assoc., 199,1043-1046 (1991)). The compound of Formula I timecourse was similar tohistoric CsA data.

In summary, the Canine KCS study demonstrated that administering thecompound of Formula I resulted in rapid improvement in Schirmer testscore in 2-8 weeks, improvement in histology, and rapidanti-inflammatory effect.

Example 7: T-Cell Proliferation Assay

This assay is an in vitro model of lymphocyte proliferation resultingfrom activation, induced by engagement of the T-cell receptor and LFA-1,upon interaction with antigen presenting cells (Springer, Nature 346:425 (1990)).

Microtiter plates (Nunc 96 well ELISA certified) are pre-coatedovernight at 4° C. with 50 μl of 2 μg/ml of goat anti-human Fc(Caltag H10700) and 50 μl of 0.07 μg/ml monoclonal antibody to CD3 (Immunotech0178) in sterile PBS. The next day coat solutions are aspirated. Platesare then washed twice with PBS and 100 μl of 17 ng/ml 5d-ICAM-1-IgG isadded for 4 hours at 37° C. Plates are washed twice with PBS prior toaddition of CD4+ T cells. Lymphocytes from peripheral blood areseparated from heparinized whole blood drawn from healthy donors. Analternative method is to obtain whole blood from healthy donors throughleukophoresis. Blood is diluted 1:1 with saline, layered and centrifugedat 2500×g for 30 minutes on LSM (6.2 g Ficoll and 9.4 g sodiumdiztrizoate per 100 ml) (Organon Technica, N.J.). Monocytes are depletedusing a myeloid cell depletion reagent method (Myeloclear, CedarlaneLabs, Hornby, Ontario, Canada). PBLs are resuspended in 90%heat-inactivated Fetal Bovine serum and 10% DMSO, aliquoted, and storedin liquid nitrogen. After thawing, cells are resuspended in RPMI 1640medium (Gibco, Grand Island, N.Y.) supplemented with 10%heat-inactivated Fetal Bovine serum (Intergen, Purchase, N.Y.), 1 mMsodium pyruvate, 3 mM L-glutamine, 1 mM nonessential amino acids, 500μg/ml penicillin, 50 μg/ml streptomycin, 50 μg/ml gentamycin (Gibco).

Purification of CD4+ T cells are obtained by negative selection method(Human CD4 Cell Recovery Column Kit # CL110-5 Accurate). 100,000purified CD4+ T cells (90% purity) per microtiter plate well arecultured for 72 hours at 37° C. in 5% CO₂ in 100 ml of culture medium(RPMI 1640 (Gibco) supplemented with 10% heat inactivated FBS(Intergen), 0.1 mM non-essential amino acids, 1 nM Sodium Pyruvate, 100units/ml Penicillin, 100 μg/ml Streptomycin, 50 μg/ml Gentamicin, 10 mMHepes and 2 mM Glutamine). Inhibitors are added to the plate at theinitiation of culture. Proliferative responses in these cultures aremeasured by addition of 1 μCi/well titrated thymidine during the last 6hours before harvesting of cells. Incorporation of radioactive label ismeasured by liquid scintillation counting (Packard 96 well harvester andcounter). Results are expressed in counts per minute (cpm).

Example 8: In Vitro Mixed Lymphocyte Culture Model

The mixed lymphocyte culture model, which is an in vitro model oftransplantation (A. J. Cunningham, “Understanding Immunology,Transplantation Immunology” pages 157-159 (1978) examines the effects ofvarious LFA-1 antagonists in both the proliferative and effector arms ofthe human mixed lymphocyte response.

Isolation of Cells: Mononuclear cells from peripheral blood (PBMC) areseparated from heparanized whole blood drawn from healthy donors. Bloodis diluted 1:1 with saline, layered, and centrifuged at 2500×g for 30minutes on LSM (6.2 g Ficoll and 9.4 g sodium diztrizoate per 100 ml)(Organon Technica, N.J.). An alternative method is to obtain whole bloodfrom healthy donors through leukophoresis. PBMCs are separated as above,resuspended in 90% heat inactivated Fetal Bovine serum and 10% DMSO,aliquoted and stored in liquid nitrogen. After thawing, cells areresuspended in RPMI 1640 medium (Gibco, Grand Island, N.Y.) supplementedwith 10% heat-inactivated Fetal Bovine serum (Intergen, Purchase, N.Y.),1 mM sodium pyruvate, 3 mM L-glutamine, 1 mM nonessential amino acids,500 μg/ml penicillin, 50 μg/ml streptomycin, 50 μg/ml gentamycin(Gibco).

Mixed Lymphocyte Response (MLR): One way human mixed lymphocyte culturesare established are in 96-well flat-bottomed microtiter plates. 1.5×10⁵responder PBMCs are co-cultured with an equal number of allogeneicirradiated (3000 rads for 3 minutes, 52 seconds stimulator PBMSc in 200μl of complete medium. LFA-1 antagonists are added at the initiation ofcultures. Cultures are incubated at 37° C. in 5% CO₂ for 6 days, thenpulsed with 1 μCi/well of 3H-thymidine (6.7 Ci/mmol, NEN, Boston, Mass.)for 6 hours. Cultures are harvested on a Packard cell harvester(Packard, Canberra, Canada). [³H] TdR incorporation is measured byliquid scintillation counting. Results are expressed as counts perminute (cpm).

Example 9: T-Cell Adhesion Assay Using Jurkat Cells

The purpose of this study was to evaluate the anti-adhesive propertiesof the compound of Formula I on the attachment of Jurkat cells to ICAM-1following in vitro exposure.

Stock solutions of the compound of Formula I and positive control wereprepared in DMSO/water (1:1) and diluted into assay media and subsequentdilutions were prepared by addition of assay media to achieve thedesired concentration. A reported LFA-1 antagonist was used as thepositive control.

Jurkat cells were labeled with an 8 μM solution of BCECF-AM(2′,7′-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein) in growthmedia at room temperature for 15 minutes. Labeled cells were incubatedin 70 μL of assay media in each well of a 96 well plate at 500,000 cellsper well with 70 μL of the compound of Formula I or positive control inassay media at 37° C. for 30 minutes. A 100 μL aliquot of thisfluorescently labeled Jurkat cell suspension was allowed to settle inthe presence of the compound of Formula I or the positive control inwells of a 96 well plate coated with recombinant human ICAM-1 expressedas an Fc chimera at 37° C. for 1 hour. Non-adherent cells were removedby washing and centrifugation at 100 g for 1 minute. Adherent cells weredetermined as adherent fluorescent units on a fluorescent plate reader.The test article, the compound of Formula I, demonstrated inhibition ofJurkat cell attachment with increasing dose. The dose response curve andIC₃₀ of the compound of Formula I in this assay was comparable to thatof the known direct competitive LFA-1 antagonist. This demonstrates thecompound of Formula I is an antagonist of LFA-1/ICAM-1 binding.

Example 10: Murine Pseudomonis Corneal Keratitis

The cornea is normally clear and leukocyte free. Bacterial infectioninduces complement mediated leukocyte recruitment and inflammation intothe cornea. A murine model of neutrophil keratitis has been developedwhich inserts a defined number of tobramycin killed Pseudomonas into asurgical cut in the cornea. Neutrophil influx and corneal haze arescored at 24 hours. The system provides a pharmacodynamic model ofneutrophil adhesion in vasculature and migration into tissue. The systemhas been described in Sun Y. and Pearlman E. (2009) Invest OphthalmolVis Sci. 50:1247-54.

Example 11: Preclinical and Clinical Safety and Tolerability: pk andSystemic and Local Distribution Results A. Effects in Humans

1. Phase 1 Clinical Trial Using the Compound of Formula I

A Phase 1 single center randomized, prospective, double masked, placebocontrolled study of escalating doses of topical compound of Formula IOphthalmic Solution was conducted in 4 cohorts (0.1%, 0.3%, 1% and 5%compound of Formula I dose strengths) in 28 healthy adults (7 subjectsper cohort: 5 received compound of Formula I Ophthalmic Solution and 2received placebo solution). The objectives of the trial were to measuresafety and tolerability, and pharmacokinetics in tear and plasma. Thedosing schedule (OU; Oculus Uterque (each eye or both eyes)) was dividedinto 3 periods, each separated by a 72-hour wash out interval:once/day×1 day (drug one eye; placebo fellow eye), twice/day×10 days,and thrice/day×10 days, 14-day observation. Slit lamp examination of theeye, BCVA (Best Corrected Visual Acuity), STTs (Schirmer Tear Test),TBUT (Tear Break-Up Time), IOP (Intraocular pressure) were assessed atscreening and the beginning and end of each period. For each cohort,masked safety data was reviewed by a Safety Committee prior to allowingdose escalation of the next cohort. A total of 2856 doses (102drops/subject) were administered over 1148 total subject study days (41study days/subject) in 56 eyes. All subjects in all cohorts completedthe study, and no study drug doses were missed.

No deaths, discontinuations, serious or severe ocular or non-ocular AEs(adverse effects) considered related to the compound of Formula IOphthalmic Solution administration occurred at any dose strength or inany dose regimen.

Blood pressure, heart rate, respiratory rate, temperature, body weight,and EKG results were within normal ranges throughout the trial.

All hematologic results and all but one serum chemistry result werewithin normal ranges with no observable study drug-related trendsmeasured across study duration, dose-strength, or schedule. Totallymphocyte count, CD3, CD4, and CD8 cell counts were within normalranges with no evidence of lymphocyte or neutrophil suppression.Urinalysis results were unremarkable throughout the trial.

Serum chemistry results were within normal range with no observablestudy drug-related trends measured across study duration, dose-strength,or schedule.

No serious or severe ocular or non-ocular AEs occurred during the study;there were 38 ocular (N=11 subjects) and 21 non ocular (N=11 subjects)AEs, respectively. There were no trends in the frequency of ocular AEswhen analyzed by dose group or by study period. No significant safetytrends were noted on BCVA, slit-lamp biomicroscopy, STT, TBUT, or IOPassessments, nor was there evidence of ocular infection, or localizedimmunosuppression. There was no evidence of localized ocular irritationor infection.

There were no trends in the frequency of non-ocular AEs when analyzed bydose group or by study period. No significant safety trends were notedon vital signs, EKG, laboratory studies (chemistry, liver functions,blood panels); there was no evidence of CD3, CD4, or CD8 T-cellsuppression, bone marrow suppression, or clinical evidence of increasedinfections.

2. Pharmacokinetics in Tear and Plasma

Plasma and tear samples were obtained at baseline and during scheduledintervals in each dosing period to characterize the pharmacokinetics(PK) of the compound of Formula I Ophthalmic Solution following ocularadministration.

a. Plasma PK Analysis

Samples for plasma compound of Formula I analysis were obtainedpre-dose, at 5 and 30 minutes post-dose, and at 1, 4, 8, 24 hourspost-dose on Days 1, 5, 14, 18 and 27. Samples were also obtained at 48hours post dose on Days 1, 14 and 27 and a single blood sample wascollected at the follow-up visit at the end of the study. Plasmacompound of Formula I concentrations were determined using a validatedLC/MS/MS (liquid chromatography tandem mass spectrometry) method with aLLOQ (Lower Limit of quantitation) of 0.500 ng/mL.

b. Plasma PK Results

The compound of Formula I plasma concentrations were BLOQ (Below thelimits of quantitation, <0.500 ng/mL) at all timepoints followingsingle- and multiple-doses of 0.1% and 0.3% the compound of Formula Idose strengths and in 3 of 5 subjects that received the 1% compound ofFormula I dose strength. Measurable levels of the compound of Formula Iwere seen in the plasma of one subject dosed with 1% compound of FormulaI at the earliest timepoint (5 minutes post-dose) on Days 14 and 27 butwere BLOQ for subsequent timepoints. Measurable levels were observedmore frequently following administration of the 5% dose strengththroughout the trial, although levels were quite low (<3 ng/mL) andgenerally were not detectable after the first hour followingadministration (FIG. 31). LFA-1 levels in in vitro cell assays (cellattachment and SEB IL-2 release) where IC50 values of 2 nM have beenobserved are approximately 0.1 nM. LFA-1 levels in blood areapproximately 10 nM. The IC50 for compound of Formula I inhibition ofSEB stimulated L-2 release in whole human blood is 69 nM. Compound ofFormula I levels greater than LFA-1 levels are needed to inhibitleukocyte function. Therefore, no significant inhibition of systemicleukocytes is expected from compound of Formula I ophthalmic drops.

Plasma compound of Formula I half-life or exposure parameters could notbe accurately assessed following administration of the compound ofFormula I Ophthalmic Solution at any dose strength in any study periodbecause the plasma compound of Formula I concentrations were notdetectable or rapidly declined BLOQ within 1 to 4 hours of dosing.

c. Tear PK Analysis

Tear samples of the compound of Formula I were collected in both eyespre-dose, at 30 minutes post-dose and at 1, 4, 8, and 24 hours post-doseon Days 1, 5, 14, 18, and 27 of the Phase 1 study using paper Schirmertear strips. A 48-hour post-dose sample was obtained following Day 1,14, and 27. Tear compound of Formula I concentrations were determinedusing a validated LC/MS/MS method with a LLOQ of 0.500 ng/mL.

d. Tear PK Results

Dose related increases in tear AUC (area under the concentration-timecurve) and C_(max) (maximum observed plasma concentration) values wereseen on dosing day 1 and were generally maintained at the timepointsevaluated throughout the trial BID (two times daily) and TID (threetimes daily) dosing produced higher C, and AUC values relative to asingle dose, but there were no significant differences in exposurebetween BID and TID dose schedules. There was clear evidence of compoundof Formula I exposure in the anticipated therapeutic dose range and noobvious evidence of accumulation with multiple ocular doseadministration.

FIG. 32 illustrates 1% compound of Formula I tear C_(min) levels. FIG.33 illustrates that dose was proportional to the compound of Formula IC_(max) tear levels. FIG. 34 illustrates that dose was proportional tothe compound of Formula I QD AUC and C_(max) in tears.

Overall, compound of Formula I Ophthalmic Solution administered bytopical ocular instillation to healthy adult subjects at dose strengthsup to 5% TID appears safe and well-tolerated and appropriate for furtherinvestigation in subjects with ocular inflammation secondary to allergicconjunctivitis or dry eye.

B. Safety Pharmacology and Toxicology Studies

1. Preclinical Toxicology Formulation

TABLE 20 Phosphate buffered saline pH 7 290 mOsM/l compound of Formula Isodium salt 4 dose levels (0.1% to 3%) EDTA Parabens preservative 0.02%methyl parabens 0.04% propyl parabens Multidose dropper bottle

2. Safety Pharmacology

An in vitro study to evaluate the effects of the compound of Formula Ion hERG channel current (a surrogate for I_(K), the rapidly activating,delayed rectifier cardiac potassium current) was conducted in stablytransfected kidney HEK293 cells. Single doses of the compound of FormulaI were 20 μM, 100 μM, 200 μM, and 600 μM. Compound of Formula I effectson the current were weak (IC₅₀ of 478 μM) indicating minimal risk ofI_(Kr) channel inhibition given the low systemic exposure observedfollowing topical ocular administration.

The cardiovascular effects of the compound of Formula I in conscioustelemetry-instrumented dogs (beagles) when administered via IV bolusinjection were assessed. No effects on electrocardiography orhemodynamic parameters were observed.

The effects of the compound of Formula I on the CNS when administered asa single dose via bolus IV injection were assessed in rats. Transientmiosis was observed in animals given 10.0 mg/kg from 1 minute to 6 hourspostdose in 2/6 animals at each time point. No effect on any otherparameters was observed.

Respiratory function (tidal volume, respiration rate, and minute volume)in rats following a single IV bolus dose of the compound of Formula Iusing head-out plethysomograph chambers was assessed. No adverse changesin respiratory function or adverse effects were observed at any dose.

3. Genotoxicity Studies:

the compound of Formula I displayed no effect in in vitro Ameschromosomal aberration assays or an in vivo rat micronucleus study.

a. In Vitro Ames Bacterial Reverse Mutation Assay

In an Ames assay, the compound of Formula I did not cause an increase inthe mean number of revertants per plate with any of the tester strainseither in the presence or absence of microsomal (S9) enzymes. Therefore,the compound of Formula I was judged to be not mutagenic.

b. In Vitro Chromosomal Aberration Assay in CHO Cells

The ability of the compound of Formula I to induce chromosomalaberrations was assessed in cultured Chinese hamster ovary (CHO) cellswith and without an exogenous metabolic activation following 20 hours ofco-incubation. The compound of Formula I is considered negative forinducing structural chromosomal aberrations in CHO cells with andwithout metabolic activation, except at a single toxic dose withoutmetabolic activation (3-hour treatment; 3500 μg/mL). The biologicalrelevance of this response is equivocal due to cytotoxicity.

c. In Vivo Mouse Bone Marrow Micronucleus Assay

The ability of repeated IV administrations of the compound of Formula Ito induce in vivo clastogenic activity and/or disruption of the mitoticapparatus, by detecting micronuclei in polychromatic erythrocytes (PCE),was assessed in CD-1® (ICR) BR mice by evaluating their bone marrow.Based on the results of this study, the compound of Formula I isconsidered negative in the mouse bone marrow micronucleus assay.

4. Acute Toxicity Studies:

For single dose IV in rats, the no observable adverse effect level(NOAEL) was 10 mg/kg IV. For escalating single dose IV and 7-dayrepeated dose with TK (toxicokinetics) in dogs, the NOAEL was 10 mg/kgIV. For single dose ocular tolerance in rabbits, the NOAEL was 3.5mg/eye/3× per day (10%).

5. Repeated Dose Toxicity Studies:

In a 4-week IV toxicity study in dogs with 2-week recovery, the NOAELwas 10 mg/kg. In a 13-week TV toxicity study in rats with 4-weekrecovery, the NOAEL was 30 mg/kg. In a 13-week ocular toxicity study inrabbits with a 4-week recovery, the NOAEL was 1.05 mg/eye/3× per day(3%). In a 13-week ocular toxicity study in dogs with a 4-week recovery,the NOAEL was 1.05 mg/eye/3× per day (3%).

6. ADME Studies

The absorption, distribution, metabolism and excretion (ADME) of thecompound of Formula I was characterized in studies conducted in rats,rabbits and dogs utilizing two routes of administration; intravenous andtopical ocular administration, the clinical route of administration. Anin vitro hepatocyte study was also performed.

The compound of Formula I levels were assessed in plasma, tear andvitreous humor samples by tandem mass spectrometry. Some in vivo studiesused [¹⁴C]-compound of Formula I to determine PK and the extent ofabsorption, distribution, and excretion of [¹⁴C]-compound of Formula Iderived radioactivity. Additionally, the metabolic profile andidentification of metabolites of [C]-compound of Formula I weredetermined in plasma, urine and feces.

Single dose ocular and IV dose administration ADME studies wereconducted in pigmented (Long-Evans strain) and albino (Sprague Dawleystrain) rats using [¹⁴C]-compound of Formula I. Quantitative whole bodyradiography assessments were performed.

Male and female rats received a single dose of 1 mg/eye or 10 mg/kg IV[¹⁴C]-compound of Formula I. The main route of excretion followingeither ocular or IV administration was the feces, accounting forapproximately 60% (ocular administration) and 95% (IV administration) ofthe administered radioactivity over 0 to 168 hours postdose. Urinaryexcretion accounted for up to 2% of the administered radioactivity. Thehighest tissue levels of [¹⁴C]-compound of Formula I were measured inthe tissues of the gastrointestinal tract with either ocular or IVdosing. With ocular administration, [¹⁴C]-compound of Formula I was alsomeasured in ocular tissues and those of excretion, indicating that theadministered dose passed from the eye through the nasal turbinates, intothe esophagus and was ultimately excreted through the gastrointestinaltract. These data indicate that ocular, nasal, or oral administration ofthe compound of Formula I will result in ultimate excretion through thegastrointestinal tract. A significant proportion of drug doseadministered as ocular drops, distributed locally to the periocularregion, and more interestingly via nasal turbinates into thegastrointestinal tract. Drug is seen to accumulate first in theepithelium of the GI tract and pass into the liver via the portal vein,where it is eliminated from the liver and re-delivered back to the lowerGI tract. Little or no drug is observed in systemic distribution.Therefore, for administration of the compound of Formula I via eitheraerosol or drops to the nose, or via oral administration may providesimilar specific direct localized delivery to the epithelium of theupper GI and localized delivery to the lower GI via clearance throughthe liver. In both cases, little or no systemic delivery of drug may bedelivered.

Following a topical ocular dose of [¹⁴C]-compound of Formula I to maleSprague Dawley (albino) rats, the distribution of radioactivity intotissues was limited at the first time point (0.5 hour postdose) and wasgenerally associated with the gastrointestinal tract, the tissuesassociated with metabolism, and the eye. FIG. 35 illustrates a wholebody autoradiograph for a male Sprague Dawley Animal 0.5 hour after asingle topical ocular administration of [¹⁴C]-compound of Formula I (1mg/eye). The highest concentrations of radioactivity were determined atthis time point in esophageal contents, nasal turbinates, and smallintestinal contents, with concentrations of 399000, 352000, and 349000ng equivalents [¹⁴C]-compound of Formula I/g, respectively. However, itshould be noted that the measurements in these tissues were above theupper limit of quantification and therefore should be interpreted withsome caution. High levels of radioactivity were also determined in theesophagus and stomach contents. Radioactivity was detected in the eye atthis time point, with a concentration of 18100 ng equivalents[¹⁴C]-compound of Formula I/g. Low levels of radioactivity were alsoassociated with the liver (272 ng equivalents [¹⁴C]-compound of FormulaI/g), kidney (151 ng equivalents [¹⁴C]-compound of Formula I/g) anduveal tract (9330 ng equivalents [¹⁴C]-compound of Formula I/g).

Concentrations of radioactivity in the eye and eye lens had declinedconsiderably by 2 hours postdose; with the level in the eye lens BLQ.Radioactivity concentrations had also declined in the esophagus andesophageal contents by approximately 50- and 100-fold at 2 hourspostdose. FIG. 36 illustrates a whole-body autoradiograph for a maleSprague Dawley Animal 2 hours after a single topical ocularadministration of [¹⁴C]-compound of Formula I (1 mg/eye). At 8 hourspostdose level of radioactivity had fallen in all tissues; however, highconcentrations were associated with the large intestinal contents(133000 ng equivalents [¹⁴C]-compound of Formula I/g) and cecum contents(57600 ng equivalents [¹⁴C]-compound of Formula I/g), indicating thepassage of radioactivity through the gastrointestinal tract. FIG. 37illustrates a whole-body autoradiograph for a male Sprague Dawley Animal8 hours after a single topical ocular administration of [¹⁴C]-compoundof Formula I (1 mg/eye).

By 12 hours postdose radioactivity concentrations had decreased further,the maximal concentrations being associated with the cecum and largeintestinal contents. The concentration determined in the uveal tractincreased at this time point to 610 ng equivalents [¹⁴C]-compound ofFormula I/g. FIG. 38 illustrates a whole-body autoradiograph for a maleSprague Dawley Animal 12 hours after a single topical ocularadministration of [¹⁴C]-compound of Formula I (1 mg/eye).

Radioactivity concentrations at 24 hours postdose were maximal in thececum contents (5870 ng equivalents [¹⁴C]-compound of Formula I/g) andthe large intestinal contents (18000 ng equivalents [¹⁴C]-compound ofFormula I/g); low levels were also present in the small intestinal andstomach contents. FIG. 39 illustrates a whole-body autoradiograph for amale Sprague Dawley Animal 24 hours after a single topical ocularadministration of [¹⁴C]-compound of Formula I (1 mg/eye). For all othertissues, with the exception of the non-pigmented skin and the liverradioactivity was not detectable.

Low levels of [¹⁴C]-compound of Formula I were measured in the vitreoushumor at all timepoints following ocular dosing and up to 2 hrsfollowing an IV dose (see schematic in FIG. 40 and Table 21 for oculardosing in rats).

TABLE 21 Compound of Formula I Concentration, ng Equivalents[¹⁴C]-compound of Formula I/g tissue. 0.5 hour after 4.0 hours afterPhysical region administration administration Aqueous humor 1770 116Conjunctiva (bulbar) 31500 4480 Conjunctiva (palpebral) 26300 21830Cornea 17150 1346 Iris-ciliary body 17550 500 Lens 38.8 9.69 Optic Nerve796 0 Retina and Choroid (with RPE) 510 46.7 Sclera 2750 387 VitreousHumor 1330 183

Tissue distribution of [¹⁴C]-compound of Formula I in pigmented andalbino rats was comparable and indicated that compound of Formula I didnot preferentially bind to melanin. There were no obvious differencesseen in results from male and female rats. Furthermore, no preferentialdistribution of [¹⁴C]-compound of Formula I-derived radioactivity wasseen in red blood cells and no metabolites were isolated from samples ofpooled plasma, urine and fecal homogenates collected up to 168 hrsfollowing either a topical ocular or IV dose administration of[¹⁴C]-compound of Formula I.

Similar single dose studies using [¹⁴C]-compound of Formula I utilizingthe same routes of administration were conducted in male and female dogs(3 mg/eye or 3 mg/dog) and showed comparable patterns of excretion,distribution and metabolism as rats. Following an ocular dose, thehighest average [¹⁴C]-compound of Formula I levels were detected inanterior ocular tissues (see FIG. 41). Lower levels were detected inposterior ocular tissues, indicating that absorption into the eye hadoccurred. The metabolic profile in pooled plasma, urine and fecalhomogenate samples was comparable to that seen in rats, with nometabolites detected up to 168 hrs post-dose. No differences in resultsfrom male and female dogs were observed.

The compound of Formula I levels in conjunctiva/cornea are greater than1 micromolar/100 nanomolar for 16 hrs (dog/rat).

a. Compound of Formula I Pharmacokinetics after Single and Repeated IVAdministration

Plasma compound of Formula I concentrations over time following a singleIV doses in rats and dogs are shown in FIGS. 42 and 43, respectively.Plasma concentrations of the compound of Formula I declined in anexpected, exponential manner following a single IV bolus dose in bothspecies.

The plasma PK parameters determined using standard noncompartmentalmethods after a single IV administration of the compound of Formula I torats at doses ranging from 0.2 to 30.0 mg/kg or to dogs after singledoses up to 30 mg/kg and 7 daily doses of 3 or 10 mg/kg are shown inTable 22 (rats). PK results from both species show very high clearanceof the compound of Formula I (liver blood flow is ˜3.3 L/hr/kg and 1.9L/hr/kg in rats and dogs, respectively; (Davies, 1993, Pharm Res). RatPK data indicated a high distribution volume, and moderate half-lifefollowing a single IV dose while low distribution volume and a shorterhalf-life drug was seen following IV administration to dogs. There wasno obvious accumulation of the compound of Formula I in plasma afterdaily administration of the compound of Formula I for 7 days as plasmacompound of Formula I C_(max) and AUC_(0-n) values measured on Study Day1 approximated those obtained on Study Day 7.

TABLE 22 Summary of Plasma PK Parameters Rats Following a Single IVBolus Dose of the Compound of Formula I³ CL Vss T_(1/2) MRT C_(max)AUC_(0-n) Dose L/hr/kg L/kg hr hr ng/mL¹ hr × ng/mL² 10.0 mg/kg 10.49.56 3.76 0.920 1056 728 30.0 mg/kg⁴ — — — — 5117.3 2345.5 ¹Maximumobserved plasma compound of Formula I concentration estimated from themean concentration versus time profile. ²Plasma compound of Formula 1AUC_(0-n) during the dose interval estimated from the mean concentrationversus time profile. ³Estimated from mean plasma compound of Formula Iconcentration versus time profiles. ⁴From rat safety pharmacology study

In longer term repeated-dose studies, dogs and rats received daily IVbolus doses of 3, 10 or 30 mg/kg/day for 4 and 13 weeks, respectively.As was seen in the 7-day dog study, plasma compound of Formula Iconcentrations declined in an expected, exponential manner and there wasno evidence of compound of Formula I accumulation in the plasma. Theplasma clearance, distribution volume, and half-life of compound ofFormula I in dogs were dose-dependent over the dose range of 3 mg/kg to30 mg/kg. In rats, the plasma compound of Formula I exposure datasuggested nonlinear disposition of compound of Formula I following dailyIV doses ranging from 10 to 30 mg/kg and unexpected accumulation at Week13 (Table 23).

TABLE 23 Plasma compound of Formula I Exposure Parameters in RatsFollowing Daily IV Bolus Doses for 13 Weeks³ Dose = Dose = 3 mg/kg Dose= 10 mg/kg 30 mg/kg AUC_(0-n) AUC_(0-n) AUC_(0-n) C_(max) hr × C_(max)hr × C_(max) hr × ng/mL¹ ng/mL² ng/mL¹ ng/mL² ng/mL¹ ng/mL² Day 1 305.2148.3 1045.3 535.6 5117.3 2345.5 Week 13 377.5 241.4 1691.5 907.116932.8 7471.5 ¹Maximum observed plasma compound of Formula Iconcentration during the dose interval. ²Plasma compound of Formula IAUC_(0-n) during the dose interval. ³Estimated and from mean plasmacompound of Formula I concentration versus time profile, n = 6 rats (3males and 3 females) per timepoint.

b. Compound of Formula I Pharmacokinetics after Single and RepeatedOcular Administration

After a single topical ocular instillation of a 0.1, 1.0 or 3.0% dosestrength of compound of Formula I Ophthalmic Solution (0.105, 0.35 and1.05 mg/eye, respectively), mean tear compound of Formula Iconcentrations rose in a dose-related manner achieving maximal valueswithin 30 minutes of administration and returning to baseline by 4hours. The tear C_(max) and AUC_(0-n) of compound of Formula I generallyincreased with increasing dose. FIG. 44 illustrates that the dose ofcompound of Formula I is proportional to PK in tears (AUC) for dogs. Forexample, mean tear C_(max) values were 34,014 ng/mL, 21460 ng/mL and313,906 ng/mL in the right eyes of rabbits dosed with 0.105, 0.35 and1.05 mg/eye, respectively. Mean tear AUCs were 18864 hr×ng/mL, 18931hr×ng/mL and 182978 hr×ng/mL in the right eyes from the same dosegroups, respectively.

Plasma compound of Formula I concentrations rose after topical ocularinstillation as the drug moved from the ocular application site into theplasma circulation. Dose-related amounts of compound of Formula I weredetected in the plasma of dogs and rabbits 30 minutes following topicalocular administration. Plasma compound of Formula I concentrationsrapidly declined from maximum values measured at about 0.25 hrspost-dose to baseline levels by about 4 hours, probably owing to thehigh compound of Formula I plasma clearance as seen in the IVadministration studies. Plasma C_(max) (mean±SD) values were 11.7±8.80ng/mL, 13.1±2.12 ng/mL, and 38.9±19.7 ng/mL and AUC_(0-n) (mean±SD)values were 5.19±539 hr×ng/mL, 735±1.52 hr×ng/mL, and 22.9±10.1 hr×ng/mLin the 0.105, 0.35, and 1.05 mg/eye/dose groups, respectively.

In repeated dose studies conducted in rabbits and dogs, compound ofFormula I Ophthalmic Solution was administered TID by bilateral ocularinstillation at the same doses as for single dose studies for 13 weeks.A pilot study in dogs administered 3.5 mg/eye (10% dose strength) for 3days. The C_(max) and AUC_(0-n) of compound of Formula I in tear samplesincreased expectedly with increasing dose in rabbits and dogs. TheC_(max) and AUC_(0-n) data indicate that compound of Formula Iaccumulated in dog tears by Week 9 during TID instillation, butthereafter continued accumulation was not noted. A similar pattern wasobserved in the rabbit study. Representative tear concentration overtime profiles measured after 13 weeks of TID ocular dosing in rabbitsand dogs are shown in FIGS. 45 and 46, respectively (left eye, TID, −4hours apart). TK (toxicokinetics) analyses indicate adequate ocularcompound of Formula I exposure with tear levels above 1 μM (600 ng/mL)throughout the day. FIG. 47 illustrates mean compound of Formula I tearconcentrations in right and left eyes of rabbits following topicalinstillation of a single dose.

Compound of Formula I was not detected in the vitreous fluid in both13-week rabbit and dog studies in samples obtained at sacrifice(terminal and recovery phase sacrifices). Variable levels of compound ofFormula I were seen in the vitreous fluid of dogs dosed TID for threedays with 3.5 mg/eye (10%) and ranged from BLOQ to 18 ng/mL.

Nonclinical studies showed that about 6.9 to 32% of the compound ofFormula I ocular dose was absorbed from the ocular topical instillationsite into the systemic circulation but this systemic availabilityestimate has been based on limited available data which includes anocular dose that is 1/100^(th) the intravenous dose. Low systemic plasmaexposure to the drug was observed in animals after ocular instillation.Importantly, the compound of Formula I plasma clearance is high in thesespecies indicating that the absorbed compound of Formula I isefficiently removed from the systemic circulation, thereby assisting tominimize systemic exposure.

The PK profiles from all nonclinical species support a clinical dosetopical ocular instillation regimen of up to three times per day for atleast 13 weeks.

c. Pilot Ocular Tolerance of Topically Administered Compound of FormulaI in Dogs-PK

A pilot ocular tolerance of topically administered compound of Formula Iin dogs-PK was performed. Animals were dosed with 35 μL of compound ofFormula I TID (0, 4, 8 hrs). 1% solution was administered on days 1-14;3% solution was administered on Days 17-21, and 10% solution wasadministered on Days 24-27. Compound of Formula I trough levels intear/periocular tissue are greater than 1000 times the IC₅₀ for T-cellattachment/IL-2 release. The compound of Formula I is safe and welltolerated at up to 10% strength at 3 doses/day. Dose dependent increasesin compound of Formula I concentration were detected in tear (30 min-16hours) and plasma (30 min) following ocular instillation. Vitreousconcentrations of compound of Formula I were greater than 1000 foldlower.

C. Dermal

1. Compound of Formula I Preclinical Dermal Studies

The compound of Formula I displays 2% (w/w) solubility inwater/glycol/transcutol solution and 10% (w/w) solubility inethanol/glycol/transcutol solution. Solubility studies suggest anemulsion formulation. Prototypes have been developed and tested onmicrotomed human skin from elective surgery at 1% (w/w). The formsinclude gels, ointment, or lotion. Stability and compatibility has beendemonstrated in all formulations. Skin transport studies performed withLC/MS/MS analysis indicate high compound of Formula I levels inepidermis and dermis and low levels in the receiver. There can begreater than 10 micromolar compound of Formula I in dermis, with 2-4%dose penetration, as determined using [¹⁴C]-compound of Formula I. Pilotrat and mini-pig studies demonstrate low systemic exposure whichindicates drug penetration into vascularized levels of skin (i.e.dermis).

2. Nonclinical Dermal Program

Dermal Sensitization Study in Guinea-Pigs: Buchler Test

A Buehler test using healthy, young adult (4 to 6 weeks), randomly bredalbino guinea pigs (strain Crl:(Ha)BR) is used to determine thepotential of compound of Formula I to induce hypersensitivity. The dietconsists of certified guinea pig diet (#5026, PMI NutritionInternational LLC) ad libitum. Water is administered ad libitum. Roomtemperature is 18 to 26° C., relative humidity is 30 to 70%, and a12-hour light/12-hour dark cycle is used. Animals are acclimated for atleast 5 days.

Experimental Design:

34 acclimated animals are placed in an irritation screening group of 4guinea pigs, a test group of 10 guinea pigs (Group 1), a naive controlgroup of 5 guinea pigs (Group 2), 10 positive control guinea pigs (Group3), and 5 positive naive control guinea pigs (Group 4).

Irritation Screen:

Hair from the back of 4 animals is removed by clipping and fourapplication sites per animal are selected. Each site is treated with 0.4mL of 0.1%, 1%, or 10% w/v compound of Formula I and 0.4-g dose ofcompound of Formula I. Appropriate concentrations of compound of FormulaI are selected for induction exposure (highest to cause mild-to-moderateskin irritation) and challenge exposure (highest non-irritant dose).

Definitive Phase:

Prior to the test, hair is removed using electric clippers from animalsin Group 1. Occlusive patch systems (Hill Top Chamber®, 25-mm diameter)are saturated with 0.4 mL solution of vehicle with a concentration ofcompound of Formula I as determined in the irritation screen. Theocclusive patches are applied to the flanks of Group 1 guinea pigs for 6hours. Restraints are used to maintain even pressure over the patches.The procedure is repeated on days 6-8 and 13-15 after the initialexposure. The positive control material, HCA(alpha-hexylcinnamaldehyde), 2.5% w/v in ethanol, is applied in asimilar manner to the Group 3 guinea pigs. The naive control animals(Groups 2 and 4) are not treated during the induction phase.

Two weeks after the last induction patch, animals are challenged withpatches saturated with a nonirritating concentration of compound ofFormula I applied to the dorsal anterior right quadrant, and along thedorsal anterior left quadrant with a challenge application of water.Group 2 animals (naive control) are shaved with electric clippers andtreated on the dorsal anterior right quadrant with compound of formula Iand along the dorsal anterior left quadrant with vehicle. HCA isadministered at 5.0% and 7.0% w/v in acetone on two respective challengesites along the right side of each animal in Group 3 in the same manneras the induction phase (0.4 mL dose volume). Group 4 animals are treatedwith two challenge applications of the positive control material in thesame manner as Group 3.

After 6 hr, the patches are removed and the area depilated (by applyingNair®). Test sites are evaluated visually 24 and 48 hr after patchremoval. Animals developing erythematous responses are consideredsensitized (if irritant control animals do not respond). The number ofpositive reactions and the average intensity of the responses arecalculated. Reactions to the challenge doses determine thesensitization. Grades of 1 or greater in the test animals to arespective material indicates evidence of sensitization, provided thatgrades of less than one are seen in the naive control animals to thissame material. If grades of one or greater are noted in the naivecontrol animals, then the reactions of test animals exceeding the mostsevere naive control reactions are considered sensitization reactions.

3. Compound of Formula I Pilot Rat Dermal Study

The safety and tolerability of prototypical dermal formulations (1%lotion, ointment, and gel) were assessed on rats given TID for sevenconsecutive days—approximately 6 cm² with 10 mg/cm². 1% DMSO was givenas a high bioavailability control. FIG. 48 illustrates that the compoundof Formula I is detectable in serum.

4. Compound of Formula I Pilot Mini-Pig Dermal Study

The tolerability and systemic exposure of various formulations of thecompound of Formula I (DMSO, gel, ointment, lotion at 1%) was assessedby giving these formulations to mini-pigs as multiple dermal does TIDfor 7 days, approximately 50 cm² with 10 mg/cm². One pig/doseformulation was used. In-life PK analysis was completed. No toxicity wasreported with any formulation. Plasma PK revealed low levels of thecompound of Formula I in all groups but below the LLOQ of 0.5 ng/ml.

The rat and mini-pig pilot studies indicate that PK were comparable withgel and ointment and the compound of Formula I is safe for evaluation inhumans as a gel or ointment formulation.

Prototypical 1% topical derm formulations have been developed (lotion,gel and ointment). There is good delivery of the compound of Formula Ito epidermis and dermis in human skin Franz cell. Pilot toxicologystudies of lotion, gel, and ointment reveal the PK demonstrates goodbioavailability.

Example 12: Phase 2 Trial Allergic Conjunctivitis

Subjects with positive history of ocular allergies and a positive skintest reaction to cat hair, cat dander, dog dander, grasses, ragweed,trees, dust mites, and/or cockroaches within the past 24 months (asdemonstrated by positive skin tests) will be challenged with allergenadministered to the conjunctiva to induce ocular itching andconjunctival redness. Subjects will be treated with both preserved andunpreserved formulations of the compound of Formula I ophthalmic drops.Unpreserved drug will be supplied as a sterile unit dose in a single useblow-fill-seal container containing the compound of Formula I formulatedin PBS. Preserved drug will be supplied as a sterile multi-use containercontaining the compound of Formula I formulated in PBS containingpreservative. Each group of test subjects will be treated QD, BID or TIDwith different dose strengths of the compound of Formula I or placebo inpreserved or unpreserved formulations. Drug will be self administered byeach subject as a single drop to each eye once, twice or three times aday as directed. Administered dose strengths will include placebo (PBSvehicle) 0.1%, 0.3%, 1% and 5% solutions of compound of Formula I.

At enrollment, subjects will be evaluated for sensitivity to allergenusing a conjunctival provocation test (also referred to as a“conjunctival allergen challenge test”). Patients responding withitchiness and redness of at least 2.0 [0-4 point scale with 0.5 pointincrements] will be supplied with drug and required to record theadministration of each drug dose in patient diaries. Patient response toallergen (itchiness and redness) will be assessed in follow-up visitswith subsequent challenges 6.7 days and/or 13.14 days after theirenrollment. Challenges in these visits will occur at variable times(approximately 15 minutes, 8 hours, or 24 hours) after their lastcompound of Formula I dose. Conversely, patients will be challenged withallergen and then treated with compound of Formula I at variable times(5 minutes, 10 minutes, 20 minutes, 40 minutes or 1 hour) after thechallenge. Patient exams will include assessments of safety, visualacuity, slit-lamp exam, dilated fundoscopy. A mean difference of atleast 1.0 point [0-4 point scale with 0.5 point increments] in ocularitching and hyperemia comparing compound of Formula I and vehicle isconsidered clinically meaningful when evaluated in the first 10 minutesfollowing allergen challenge.

Objective measures of efficacy (physician reported) will include: 1)conjunctival hyperemia, 2) episcleral hyperemia, 3) ciliary hyperemia,and 4) chemosis.

Subjective measures of efficacy (patient reported) will include: 1)ocular itching, 2) blepharitis, 3) rhinorrhea, 4) nasal congestion, and5) nasal pruritis.

For seasonal allergies, subjects will be treated daily at QD, BID or TIDdoses for up to 8 consecutive weeks during peak allergy season forcommon grass and tree pollens (also referred to as “environmentalstudies”). Similar measures of objective and subjective efficacymeasures will be evaluated.

For either environmental or conjunctival provocation studies, a safetytrial of at least 6 months will be conducted in normal adult andpediatric patients.

Results of this trial will support regulatory claims to the treatment orprevention of signs and symptoms from allergic conjunctivitis (bothseasonal and perennial); steroid sparing treatment of allergicconjunctivitis—no steroid safety events (glaucoma, cataracts); compoundof Formula I can be used in conjunction with mast cell stabilizers andantihistamines to enhance or prolong efficacy; treatment of both ocularand nasal signs and symptoms of allergy.

Example 13: Phase 2 Trial for Dry Eye

Subjects with moderate to severe dry eye will be treated for 12 weeks(efficacy trials) and up to 1 year (safety trials) with both preservedand unpreserved formulations of compound of Formula I ophthalmic drops.Unpreserved drug will be supplied as a sterile unit dose in a single useblow-fill-seal container containing compound of Formula I formulated inPBS. Preserved drug will be supplied as a sterile multi-use containercontaining compound of Formula I formulated in PBS containingpreservative. Each group of test subjects will be treated QD or BID withdifferent dose strengths of compound of Formula I or placebo inpreserved or unpreserved formulations. Drug will be self administered byeach subject as a single drop to each eye once or twice a day.Administered dose strengths will include placebo (PBS vehicle) 0.1%,0.3%, 1% and 5% solutions of compound of Formula I.

At enrollment, subjects will be evaluated for signs and symptoms of DryEye. Patients will be supplied with drug and required to record theadministration of each drug dose in patient diaries. Patient signs andsymptoms of Dry Eye will be assessed in follow-up visits at the end ofweek 2, week 4, week 6, week 8 and/or week 12. Patient exams willinclude assessments of safety, visual acuity, slit-lamp exam, dilatedfundoscopy. Endpoints will be measured at the clinic in normal officeconditions (referred to as “environmental” conditions) and measuredduring and/or immediately following prolonged exposure to a controlledenvironment (i.e., controlled humidity, temperature, air-flow, andvisual tasking; also referred to as a “controlled ambient environment”).

Objective clinical measures of efficacy will include: 1) cornealstaining with fluorescein, 2) conjunctival staining with lissaminegreen, 3) tear film break up time with fluorescein, 4) Schirmer teartests with and without anesthesia, 5) conjunctival impression cytology(ICAM-1), 6) tear osmolarity, 7) blink rate, 8) ocular hyperemia, 9)Cochet Bonnet corneal sensitivity, 10) tear fluorophotometry, and 11)ocular protection index.

Subjective clinical measures of efficacy will include: 1) Ocular SurfaceDisease Index, 2) Patient global self-assessment (self-scored oculardiscomfort) 3) Visual analog scale, and 4) drop comfort (tolerabilityassessment).

Results of this trial will support regulatory claims to the treatment orprevention of signs and symptoms from keratoconjunctivitis sicca (dryeye) with or without concomitant use of lubricating eye drops.

Example 14: Diabetic Retinopathy (DR) and Diabetic Macular Edema (DME)

DR and DME are leukocyte mediated diseases. Adhesion of leukocyte tocapillary epithelial cells seems critical in ischemia reperfusionmechanism.

Human Study

Subjects with type I or type II diabetes will be treated with compoundof Formula I for up to 3 years with both preserved and unpreservedformulations of compound of Formula I ophthalmic drops. Unpreserved drugwill be supplied as a sterile unit dose in a single use blow-fill-sealcontainer containing compound of Formula I formulated in PBS. Preserveddrug will be supplied as a sterile multi-use container containingcompound of Formula I formulated in PBS containing preservative. Eachgroup of test subjects will be treated QD, BID or TID with differentdose strengths of compound of Formula I ophthalmic drops or placebo inpreserved or unpreserved formulations. Drug will be self administered byeach subject as a single drop to each eye once, twice or three times aday. Administered dose strengths will include placebo (PBS vehicle)0.1%, 0.3%, 1% and 5% solutions of compound of Formula I. To enhancepatient compliance, compound of Formula I can be administered as a slowrelease formulation which delivers drug to the retina over the course ofthe study.

At enrollment, patients must have a diagnosis of type I or type IIdiabetes and non-proliferative diabetic retinopathy. Patients may alsohave concomitant diabetic macular edema. Patients will be supplied withdrug and required to record the administration of each drug dose inpatient diaries. Patients will be assessed every 2 months for theduration of the study. Each patient exam will include assessments ofsafety, visual acuity, slit-lamp exam, dilated fundoscopy.

Objective measures of efficacy will include: 1) Best corrected visualacuity using Early Treatment of Diabetic Retinopathy (ETDRS) method at 4meters, 2) Reduction in retinal thickness measured by optical coherencetomography (OCT), and 3) Progression of diabetic retinopathy.

Subjective clinical measures of efficacy will include: 1) improvementNEI-VFQ 25 and other validated patient-reported outcome instruments.

Results of this trial will support regulatory claims to the preventionof the progression of diabetic retinopathy at 4, 8 weeks, 1, 2, and 3years; maintenance or improvement in visual acuity; prevention,treatment, and/or reduction in macular edema; can be used in combinationwith focal and grid laser, intravitreal steroids, photodynamic therapy,and/or anti-VEGF therapies.

Rat STZ Model of Diabetic Macular Edema (DME) Pilot Study

Anti-ICAM antibodies have shown efficacy in a rat STZ model of DME.Compound of Formula I radiolabel distribution studies in rat demonstratedelivery to retina. STZ (strptozocin) is used to generate an animalmodel for Type 1 diabetes. A definitive STZ rat study with compound ofFormula I will include 5 groups with 18 animals. Group no. 1 is normalSD rats that will receive no treatment. Group no. 2 is STZ rats thatreceive vehicle drops BID/2 months. Group no. 3 is STZ rats that receive1% compound of Formula I drops BID/2 months. Group no. 4 is STZ ratsthat will receive 5% compound of Formula I drops BID/2 months. Group no.5 is STZ rats that will receive celecoxib positive control Endpoints forthe study will include: retinal FITC-dextran leakage, vitreous-plasmaprotein ratio, myeloperoxidase assay, and retinal leukostasis.

Leukostasis is studied as described in U.S. Patent Application No.20080019977 using Acridine Orange Leukocyte Fluorography (AOLF) andFluorescein Angiography. Leukocyte dynamics in the retina are studiedwith AOLF (Miyamoto, K., et al., Invest. Opthalmol. Vis. Sci.,392190-2194 (1998); Nishiwaki, H., et al., Invest. Opthalmol. Vis. Sci.,37:1341-1347 (1996); Miyamoto, K., et al., Invest. Opthalmol. Vis. Sci.,37:2708-2715 (1996)). Intravenous injection of acridine orange causesleukocytes and endothelial cells to fluoresce through the non-covalentbinding of the molecule to double stranded nucleic acid. When a scanninglaser opthalmoscope is utilized, retinal leukocytes within blood vesselscan be visualized in vivo. Twenty minutes after acridine orangeinjection, static leukocytes in the capillary bed can be observed.Immediately after observing and recording the static leukocytes,fluorescein angiography is performed to study the relationship betweenstatic leukocytes and retinal vasculature.

Twenty-four hours before AOLF and fluorescein angiography is performed,all rats had a heparin-lock catheter surgically implanted in the rightjugular vein for the administration of acridine orange or sodiumfluorescein dye. The catheter is subcutaneously externalized to the backof the neck. The rats are anesthetized for this procedure with xylazinehydrochloride (4 mg/kg) and ketamine hydrochloride (25 mg/kg).Immediately before AOLF, each rat is again anesthetized, and the pupilof the left eye is dilated with 1% tropicamide to observe leukocytedynamics. A focused image of the peripapillary fundus of the left eye isobtained with a scanning laser opthalmoscope (SLO). Acridine orange isdissolved in sterile saline (1.0 mg/ml) and 3 mg/kg is injected throughthe jugular vein catheter at a rate of 1 ml/min. The fundus is observedwith the SLO using the argon blue laser as the illumination source andthe standard fluorescein angiography filter in the 40° field setting for1 minute. Twenty minutes later, the fundus is again observed to evaluateleukostasis in the retina. Immediately after evaluating retinalleukostasis, 20 μl of 1% sodium fluorescein dye is injected into thejugular vein catheter. The images are recorded on a videotape at therate of 30 frames/sec. The video recordings are analyzed on a computerequipped with a video digitizer that digitizes the video image in realtime (30 frames/sec) to 640×480 pixels with an intensity resolution of256 steps. For evaluating retinal leukostasis, an observation areaaround the optic disc measuring ten disc diameters in diameter isdetermined by drawing a polygon surrounded by the adjacent major retinalvessels. The area is measured in pixels and the density of trappedleukocytes is calculated by dividing the number of trapped leukocytes,which are recognized as fluorescent dots, by the area of the observationregion. The leukocyte densities are calculated generally in eightperipapillary observation areas and an average density is obtained byaveraging the eight density values.

Compound of Formula I is expected to reduce leukostasis andblood-retinal barrier leakage in STZ treated rats.

Example 15: Age Related Macular Degeneration (AMD)

Subjects with wet or dry AMD will be treated with compound of Formula Ifor up to 3 years with both preserved and unpreserved formulations ofcompound of Formula I ophthalmic drops. Unpreserved drug will besupplied as a sterile unit dose in a single use blow-fill-seal containercontaining compound of Formula I formulated in PBS. Preserved drug willbe supplied as a sterile multi-use container containing compound ofFormula I formulated in PBS containing preservative. Each group of testsubjects will be treated QD, BID or TID with different dose strengths ofcompound of Formula I ophthalmic drops or placebo in preserved orunpreserved formulations. Drug will be self administered by each subjectas a single drop to each eye once, twice, or three times a day.Administered dose strengths will include placebo (PBS vehicle) 0.1%,0.3%, 1% and 5% solutions of compound of Formula I. To enhance patientcompliance, compound of Formula I can be administered as a slow releaseformulation which delivers drug to the retina over the course of thestudy.

At enrollment, patients must have a diagnosis of wet or dry AMD.Patients may also have concomitant diabetic macular edema. Patients willbe supplied with drug and required to record the administration of eachdrug dose in patient diaries. Patients will be assessed every 2 monthsfor the duration of the study. Each patient exam will includeassessments of safety, visual acuity, slit-lamp exam, dilatedfundoscopy.

Objective measures will include: best corrected visual acuity;prevention of progression of geographic atrophy; and prevention ofconversion to neovascular (wet AMD).

Results of this trial will support regulatory claims to the preventionof geographic atrophy related to dry AMD; can be used in conjunctionwith genetic biomarker or other type of diagnostic study that predictssubjects at high risk; and can be used in conjunction with anti-oxidantand/or anti-neovascular or anti-VEGF agents.

Example 16: Phase 2 Atopic Dermatitis

Subjects with atopic dermatitis will be treated with compound of FormulaI for up to 12 months. Drug will be supplied as a suitable dermatologicformulation for local application (cream, lotion, gel or ointment)containing compound of Formula I. Each group of test subjects will betreated QD, BID or TID with different dose strengths of compound ofFormula I ophthalmic drops or placebo in formulation. Drug will be selfadministered by each subject by gentle rubbing onto the effected area.Administered dose strengths will include placebo (vehicle) 0.1%, 0.3%,1%, and 2% preparations of compound of Formula I. To enhance effect,treated areas may covered with an occlusive dressing. To improve patientcompliance, drug may be administered as a slow release drug-impregnatedpatch.

At enrollment, patients must have a diagnosis of atopic dermatitis.Patients will be supplied with drug and required to record theadministration of each drug dose in patient diaries. Patients will beassessed every 2 weeks for the duration of the study. Each patient examwill include assessments of safety and tolerability. Measures ofefficacy will include a physician's global assessment, a reduction inaffected body surface area or a reduction in pruritis score.

Results of this trial will support regulatory claims to treatment ofatopic dermatitis.

Example 17: Crohn's Disease, Ulcerative Colitis or IBD

Subjects with Crohn's disease, ulcerative colitis or IBD will be treatedwith compound of Formula I for up to 12 months. Drug will be supplied asa formulation suitable for oral administration (solution, pill, orcapsule) containing compound of Formula I. A typical oral solutiondosage form would include compound of Formula I dissolved in PBSadjusted to pH 7. Each group of test subjects will be treated QD, BID orTID with different dose strengths of compound of Formula I or placebo informulation. Drug will be self administered by each subject by mouth.Administered dose strengths will include placebo (vehicle) 1 mg perdose, 5 mg per dose, 10 mg per dose and up to 100 mg per dose ofcompound of Formula I in formulation.

At enrollment, patients must have a diagnosis of Crohn's disease,ulcerative colitis or IBD. Patients will be supplied with drug andrequired to record the administration of each drug dose in patientdiaries. Treatment with compound of Formula I can be used in conjunctionwith current anti-infamnmatories (eg, salicylates) andimmunosuppressants (methotrexates, steroids, antibodies).

Patients will be assessed every 2 weeks for the duration of the study.Each patient exam will include assessments of safety and tolerability.Measures of efficacy will include the Crohn's Disease Activity Index(CDAI); disease activity index or similar scale for ulcerative colitis.

Results of this trial will support regulatory claims to the treatmentand maintenance of remission of Crohn's disease, ulcerative colitisand/or IBD.

While selected embodiments of the present invention have been shown anddescribed herein, it will be obvious to those skilled in the art thatsuch embodiments are provided by way of example only. Numerousvariations, changes, and substitutions will now occur to those skilledin the art without departing from the invention. It should be understoodthat various alternatives to the embodiments of the invention describedherein may be employed in practicing the invention. It is intended thatthe following claims define the scope of the invention and that methodsand structures within the scope of these claims and their equivalents becovered thereby.

1. A method of synthesizing a compound of Formula I:

comprising the steps: a) base hydrolysis of Formula AA with a base in anaprotic solvent:

wherein R is a carbon containing moiety; and b) isolating a compound ofFormula I.
 2. The method of claim 1, wherein the aprotic solvent isdioxane.
 3. The method of claim 1, wherein the base is sodium hydroxide.4. The method of claim 1, wherein R is a substituted or unsubstitutedgroup selected from lower alkyl, lower alkenyl, lower alkynyl,cyclo(lower)alkyl, cyclo(lower)alkenyl, aryl, aralkyl, heterocyclyl, andheteroaryl groups.
 5. The method of claim 1, comprising the steps: a)base hydrolysis of Formula A:

with a base in an aprotic solvent; and b) isolating a compound ofFormula I.
 6. The method of claim 5, wherein the aprotic solvent isdioxane.
 7. The method of claim 5, wherein the base is sodium hydroxide.8. A method of synthesizing a compound of Formula I:

comprising the steps: a) acid hydrolysis of Formula AA with an acid inan aprotic solvent:

wherein R is a carbon containing moiety; and b) isolating a compound ofFormula I.
 9. The method of claim 8, wherein the aprotic solvent isdioxane.
 10. The method of claim 8, wherein the acid is hydrogenchloride.
 11. The method of claim 8, wherein R is a substituted orunsubstituted group selected from lower alkyl, lower alkenyl, loweralkynyl, cyclo(lower)alkyl, cyclo(lower)alkenyl, aryl, aralkyl,heterocyclyl, and heteroaryl groups.
 12. The method of claim 8,comprising the steps: a) acid hydrolysis of Formula A:

with an acid in an aprotic solvent; and b) isolating a compound ofFormula I.
 13. The method of claim 12, wherein the aprotic solvent isdioxane.
 14. The method of claim 12, wherein the acid is hydrogenchloride.
 15. An isolated compound of Formula I:

wherein said compound is synthesized according to the method of claim 1,and further wherein said compound has a lower level of residualpalladium compared to a compound of Formula I prepared bypalladium-catalyzed transfer hydrogenolysis.